ABSTRACT
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ABSTRACT
Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.
Subject(s)
Breast Neoplasms/metabolism , Cadherins/metabolism , Epithelial-Mesenchymal Transition/physiology , Insulin-Like Growth Factor Binding Protein 6/metabolism , Breast Neoplasms/genetics , Cadherins/genetics , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/physiology , Humans , Insulin-Like Growth Factor Binding Protein 6/genetics , Models, BiologicalABSTRACT
IGFBP6 gene plays an important role in the pathogenesis of breast cancer. In this work, we performed knockdown of IGFBP6 gene in MDA-MB-231 cells and obtained a stable cell line. Knockdown of IGFBP6 gene was confirmed by the real-time PCR. The influence of IGFBP6 gene on migration and proliferation of breast cancer cells was studied. Knockdown of IGFBP6 gene reduced migration activity of MDA-MB-231 cells and increased their proliferation rate. This in vitro cell model can be used for the further analysis of the role of IGFBP6 gene in the pathogenesis of breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Insulin-Like Growth Factor Binding Proteins/metabolism , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cell Proliferation/genetics , Cell Proliferation/physiology , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Insulin-Like Growth Factor Binding Proteins/genetics , Real-Time Polymerase Chain ReactionABSTRACT
The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.
Subject(s)
Lentivirus/genetics , Mesenchymal Stem Cells/metabolism , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Female , Gene Library , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Plasmids/genetics , Plasmids/metabolism , Polymerase Chain ReactionABSTRACT
Multiple myeloma is a mostly incurable malignancy characterized by the expansion of a malignant plasma cell (PC) clone in the human bone marrow (BM). Myeloma cells closely interact with the BM stroma, which secretes soluble factors that foster myeloma progression and therapy resistance. Growth arrest-specific gene 6 (Gas6) is produced by BM-derived stroma cells and can promote malignancy. However, the role of Gas6 and its receptors Axl, Tyro3 and Mer (TAM receptors) in myeloma is unknown. We therefore investigated their expression in myeloma cell lines and in the BM of myeloma patients and healthy donors. Gas6 showed increased expression in sorted BMPCs of myeloma patients compared with healthy controls. The fraction of Mer(+) BMPCs was increased in myeloma patients in comparison with healthy controls whereas Axl and Tyro3 were not expressed by BMPCs in the majority of patients. Downregulation of Gas6 and Mer inhibited the proliferation of different myeloma cell lines, whereas knocking down Axl or Tyro3 had no effect. Inhibition of the Gas6 receptor Mer or therapeutic targeting of Gas6 by warfarin reduced myeloma burden and improved survival in a systemic model of myeloma. Thus, the Gas6-Mer axis represents a novel candidate for therapeutic intervention in this incurable malignancy.
Subject(s)
Gene Expression Regulation, Neoplastic , Intercellular Signaling Peptides and Proteins/genetics , Multiple Myeloma/genetics , Plasma Cells/metabolism , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Case-Control Studies , Cell Line, Tumor , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred NOD , Multiple Myeloma/mortality , Multiple Myeloma/pathology , Multiple Myeloma/therapy , Neoplasm Transplantation , Plasma Cells/pathology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Stromal Cells/metabolism , Stromal Cells/pathology , Survival Analysis , Warfarin/pharmacology , c-Mer Tyrosine Kinase , Axl Receptor Tyrosine KinaseABSTRACT
Biopsy specimens of the terminal duodenum obtained from 11 patients with hereditary haemochromatosis were examined by light and electron microscopy. Stainable iron was found in the lamina propria of the terminal duodenum in only 4 patients, all of whom were in an advanced stage of the disease. The iron was localized in the basal parts of the villi, sparing their tips, and between the crypts of Lieberkühn. The iron-storing cells could be identified as plasma cells, in which ferritin and haemosiderin were localized within lysosomes and ferritin molecules scattered in the cell sap. There was no storage of iron in macrophages. These observations demonstrate the impaired iron-storing capacity of macrophages in hereditary haemochromatosis, which may be related to the increased iron absorption in this iron storage disease.
