ABSTRACT
Osteopontin (OPN) is a secreted, non-collagenous, sialic acid-rich protein which functions by mediating cell-matrix interactions and cellular signaling via binding with integrins and CD44 receptors. An increasing number of studies have shown that OPN plays an important role in controlling cancer progression and metastasis. OPN was found to be expressed in many human cancer types, and in some cases, its over-expression was shown to be directly associated with poor patient prognosis. In vitro cancer cell line and animal model studies have clearly indicated that OPN can function in regulating the cell signaling that ultimately controls the oncogenic potential of various cancers. Previous studies in our laboratory demonstrated that OPN is highly expressed in human osteosarcoma (OS) cell line OS-732. In this study, we successfully reduced the tumorigenecity of OS-732 cells xenotransplanted into nude mice, using the antisense human OPN (hOPN) RNA expression vector.
Subject(s)
Osteopontin/genetics , Osteosarcoma/genetics , Osteosarcoma/therapy , RNA, Antisense/therapeutic use , Animals , Cell Line, Tumor , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Osteosarcoma/pathologyABSTRACT
AIM: To examine the role of nucleostemin in the growth regulation of gastric cancer, liver cancer and other cancers. METHODS: RT-PCR was used to clone the fragment of nucleostemin cDNA from HEK 293 cells. Eighteen kinds of malignant tumor tissues including gastric adenocarcinoma and liver cancer tissues, 3 kinds of benign tumor tissues, 3 kinds of benign hyperplastic tissues and normal tissues were employed to examine nucleostemin gene expression by RT-PCR, Slot blot, Northern blot and in situ hybridization. RESULTS: We successfully cloned a 570 bp fragment of nucleostemin-cDNA from HEK-293 cells. All detected malignant tumor tissues, benign tumor tissues, and benign hyperplastic tissues had high levels of nucleostemin expression. Nucleostemin was also expressed in human placenta tissue at a high level. In terminally differentiated normal human adult kidney and mammary gland tissues, no nucleostemin expression could be detected. CONCLUSION: Nucleostemin can help regulate the proliferation of both cancer cells and stem cells. It might play an important role in the growth regulation of gastric cancer, liver cancer and other cancers.
Subject(s)
Carrier Proteins/metabolism , Liver Neoplasms/metabolism , Nuclear Proteins/metabolism , Stomach Neoplasms/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carrier Proteins/genetics , Cell Line , Cloning, Molecular , Female , GTP-Binding Proteins , Gene Expression Regulation , Humans , In Situ Hybridization , Kidney/cytology , Kidney/metabolism , Liver Neoplasms/genetics , Nuclear Proteins/genetics , Stomach Neoplasms/genetics , Tissue DistributionABSTRACT
BACKGROUND: To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro. METHODS: The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E. coli BL21 (DE3) cells and purified with ProBond trade mark Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel. RESULTS: The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells. CONCLUSION: hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.
Subject(s)
Bone Neoplasms/pathology , Cell Division/drug effects , Cell Movement/drug effects , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Osteosarcoma/pathology , Sialoglycoproteins/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Humans , OsteopontinABSTRACT
Chromosomal DNA in higher eukaryotes is spatially organized into loops by periodic attachment to the nuclear matrix at its base via a specific matrix attachment region (MAR). In order to study the nature of DNA sequences that affixed the loops to the nuclear matrix, we have cloned the MAR DNA from bovine lactating mammary tissues. In vitro binding assay showed that the cloned fragments could be co-complexed with nuclear matrix proteins to form insoluble complex easily removed by centrifugation. Sequences of the two chosen MAR loci are composed of TG-, CA- and GA- blocks, as well as the ATTA motifs. Both the MAR loci show numerous replication/transcription factor binding sites, enhancer motifs, several perfect or imperfect inverted repeats, and sequences sharing the common features of the potential DNA bending core sequence. The possibility that a combination of different elements in the same DNA sequence may function as either positive or negative regulatory elements in controlling a variety of cellular and developmental processes is discussed.
