BACKGROUND: We have previously shown that the vimentin-positive subset of human breast cancer cell lines, which are more invasive in vitro and in vivo, can activate MMP-2 when cultured on vitrogen gels whereas the poorly invasive, poorly metastatic vimentin-negative subset cannot (J. Cell Physiol. 150: 534, 1992). METHODS: In this study, we compared the interaction of human breast cancer cell lines from each group with respect to the interaction with vitrogen. RESULTS: The cell lines capable of responding to vitrogen for MMP-2 activations showed an enhanced ability to attach to vitrogen-coated culture wells. MCF-7 (non-MMP-2 activating) and MDA-MB-231 (MMP-2 activating) cells were selected for more detailed analysis. MDA-MB-231 cells showed a greater affinity for the 3-dimensional vitrogen than MCF-7 cells. Attachment of both lines to thin coatings of vitrogen was shown to require divalent cations and to be mediated by beta1 integrins. The alpha5 subunit, however, was shown to be involved in fibronectin attachment, but not vitrogen attachment. The GRGDSP peptide dramatically inhibited fibronectin attachment of both cell lines, but did not effect the vitrogen attachment of either. In contrast, the KGDEA recognition sequence for alpha2 integrin inhibited the attachment of MDA-MB-231 cells to vitrogen, but not to fibronectin or laminin. CONCLUSIONS: These results show that the subset of human breast cancer cell lines which respond to the vitrogen gel with MMP-2 activation may interact more easily with the vitrogen, apparently through integrin-mediated recognition. Preliminary analysis reveals that the alpha2beta1 integrin, previously implicated in vitrogen interaction may mediate this response.
Antigens, CD29 ; Breast Neoplasms* ; Breast* ; Cations, Divalent ; Cell Line* ; Fibronectins ; Gels ; Humans* ; Integrin alpha2 ; Laminin ; MCF-7 Cells

Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90%. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.
Antigens, CD29/analysis ; Antigens, CD44/analysis ; Bone Marrow Cells/*cytology ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Flow Cytometry ; Mesenchymal Stem Cells/*cytology

PURPOSE: We explored the possibility of DNA methylation as a mechanism of loss of type II collagen expression in dedifferentiating chondrocytes by culturing in monolayer. MATERIALS AND METHODS: Dedifferentiation was induced by low density subculturing primary porcine chondrocytes in vitro. The mRNA expression of Type I collagen, Type II collagen and DNA methyltransferase (DNMT) was measured by RT-PCR. Induction of redifferentiation in dedifferentiated chondrocytes was performed in 3-dimensional alginate bead culture system. As stimulating factors for reexpression of genes in dedifferentiated chondrocytes, 10 ng/ml TGF-beta1 and 5 micrometer 5-azacytidine were used. RESULTS: Type II collagen mRNAs was expressed strongly in freshly isolated cells but had decreased in monolayer cultured cells after 3 weeks up to 40%. In contrast, type I collagen expression was increased from 21 days and kept increasing during the 86 days of study. After treatment of 5 micrometer 5-azacytidine, fibroblast like morphology was changed to round shape such as traditional chondrocyte morphology at day 4. At day 10, type II collagen expression was increased by 5-azacytidine and TGF-beta1 marginally and also integrin beta1 expression was increased in all groups. RT-PCR analysis demonstrated that DNMT3A expression increased in dedifferentiating chondrocytes when compared with control cells for 40 days. CONCLUSION: Loss of type II collagen mRNA expression and increase of DNMT 3A expression were showed similar patterns during dedifferentiation. These results suggest that type II collagen gene expression may be influenced by DNA methylation. As stimulating factors, TGF-beta1 and 5-azacytidine have potential activity to increase the type II collagen expression in alginate culture system.
Antigens, CD29 ; Azacitidine ; Cells, Cultured ; Chondrocytes* ; Collagen Type I ; Collagen Type II* ; DNA Methylation* ; DNA* ; Fibroblasts ; Gene Expression ; RNA, Messenger ; Transforming Growth Factor beta1

PURPOSE: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. METHODS: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. RESULTS: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin beta1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. CONCLUSION: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.
Abdominal Fat ; Adipose Tissue ; Antigens, CD29 ; Apoptosis ; Bone Marrow ; Cell Adhesion ; Cell Proliferation ; Cytoplasm ; Extracellular Matrix ; Fetal Blood ; Fibronectins ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; Microscopy ; Seeds ; Sincalide

PURPOSE: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. METHODS: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. RESULTS: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin beta1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. CONCLUSION: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.
Abdominal Fat ; Adipose Tissue ; Antigens, CD29 ; Apoptosis ; Bone Marrow ; Cell Adhesion ; Cell Proliferation ; Cytoplasm ; Extracellular Matrix ; Fetal Blood ; Fibronectins ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; Microscopy ; Seeds ; Sincalide

PURPOSE: Integrins are cell surface proteins that anchor the cells to the extra-cellular matrix. It has recently been found that integrins are involved in proliferations, migration, differentiation and survival signal transduction. We studied the expression of integrins in normal and cancer tissue of Korean breast cancer patients, and investigated the relationship between integrin expression and the characteristics of breast cancer. METHODS: Normal and malignant breast tissues were taken from 25 breast cancer patients who were admitted to the Ajou University Hospital. Specimens were immediately preserved in a nitrogen tank at the time of the operation. Total RNA was extracted, and semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCR) performed with PCR primers for integrin alpha1, alpha2, alpha5, and alphav, and integrin beta1, and beta3. The integrin expressions were compared between the normal and malignant tissues, and the expressions were analyzed in relation to tumor characteristics. RESULTS: Integrin alpha1, alpha5, alphav, beta1, and beta3 were significantly over-expressed in breast cancer tissue than in normal tissue. There was no difference in integrin alpha2 expression between the normal and cancer tissues. Integrin beta1 was over-expressed to a greater extent in lower histological grade carcinomas and to a lesser extent in high grade tumors. Hormonal receptor positive tumor tissue had more alphav, alpha5, and beta1 integrin expressions. There was no significant relationship between integrins and tumor size, lymph node meta-stasis, lymphovascular involvement, or c-erb-B2 expression. CONCLUSIONS: Integrins alpha1, alpha5, alphav and beta3 were over- expressed in malignant breast tissue to a greater extent than in normal tissue. However, studies on the localization of integrin expression in cancer tissue, and co-relations of integrin over-expressions, with survival and drug sensitivity, must be followed to evaluate the clinical value of integrin expression.
Antigens, CD29 ; Breast Neoplasms* ; Breast* ; Humans ; Integrin alpha1 ; Integrin alpha2 ; Integrins* ; Lymph Nodes ; Membrane Proteins ; Nitrogen ; Polymerase Chain Reaction ; RNA ; RNA-Directed DNA Polymerase ; Signal Transduction

OBJECTIVE: To comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue. METHODS: Degenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins alphav, beta1, and beta3 was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Immunohistochemical staining showed that vimentin and integrin alphav was broadly expressed in all tissues examined. By contrast, integrin beta1 was weakly expressed only in 38.4% (5/13) of tissues. Integrin beta3 was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin alphav in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin beta1 was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin beta3 was variable. CONCLUSION: Spinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin alphav and beta1 in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.
Adhesives ; Antigens, CD29 ; Bone and Bones ; Breast ; Breast Neoplasms ; Colonic Neoplasms ; Gene Expression ; Humans ; Immunohistochemistry ; Integrin alphaV ; Integrin beta3 ; Integrins* ; Liver Neoplasms ; Lung Neoplasms ; Neoplasm Metastasis ; Spine* ; Vimentin*

INTRODUCTION: The pathophysiology of PIH remains unclear. Recently, placental abnormalitiesare stressed as a possible cause of PIH. Abnormal shallow invasion of trophoblasts, confinedto decidua, without involving myometrium is believed to result in reduced uteroplacentalperfusion, endothelial injury, and activation of coagulation cascade system. Integrin, one of theadhesive membrane proteins, is expected to be related to the regulation of trophoblasts invasion. PURPOSE: The purpose of this study is to investigate the expression of adhesion moleculesin placenta and the correlation between uterine artery Doppler findings and integrinexpressions in the placentas of PIH patients. SUBJECTS: Thirty-six cases of severe PIH patients were enrolled in the study with 10number of normal control pregnant women. The integrin subunit expressions withimmunohistochemical staining were observed in floating villi, maternal-side cytotropholbasts, andfetal-side cytotrophoblasts. Uterine artery Doppler study was also performed, and the S/Dratio was evaluated. Abnormal Doppler findings was defined as S/D ratio>or=2.6. RESULTS: Cytoplasmic staining of villi and placental bed cytotrophoblast for theintegrin alpha1 subunit in PIH specimen was weaker than those in normal controls. Theexpression of integrin beta1 subunit was negative for both controls and PIH group. Thepositive cytoplasmic stain was observed among PIH placenta in contrast to normal control inwhich the expression of integrin beta4 subunit was not detected. The expression of alpha v beta3 introphoblast with PIH was positive staining, but not in control group. Uterine artery Dopplervelocimetry was performed in 25 cases with PIH. Trace(+/-) or - staining of integrin alpha1 subunit were observed in 60.0% of abnormal S/D(>or=2.6) group, 20.0% of normal S/Dratio group patients, respectively. Trace or + staining of integrin beta4 subunit were observedin 50.0% of abnormal S/D group and 6.7% of normal S/D group and this is in statisticallysignificant. Trace or + staining of integrin alpha v beta3 subunit were observed 70.0% ofabnormal S/D group and 26.7% of normal S/D group, and this statistically significant. CONCLUSION: In PIH the abnormality in the invasion of cytotrophoblats results inabnormal integrin subunit expression, but it is also correlated to the abnormal uterine arteryDoppler velocimetry which shows a S/D ratio of greater than 2.6. Thus, the uterine arteryDoppler velocimetry reflects abnormal placentation.
Animals ; Antigens, CD29 ; Cytoplasm ; Decidua ; Female ; Humans ; Hypertension, Pregnancy-Induced* ; Integrin alpha1 ; Integrin alphaV ; Integrin beta4 ; Integrins ; Membrane Proteins ; Mice ; Myometrium ; Placenta* ; Placentation ; Pregnancy ; Pregnant Women ; Rheology* ; Trophoblasts ; Uterine Artery*

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates the func-tions of beta1 integrin, suggesting that it may play a role in tumor cell invasion. In this study, the effects of CD98 signaling on the adhesion and invasion of tumor cells were investigated. The expression of CD98 on MCF-7 human breast carcinoma cells was confirmed by immunohistochemistry. The effects of CD98 activation on the adhesion to extracellular matrix (ECM) and invasion of MCF-7 cells were determined by adhesion assay and cell invasion assay. Dominant negative forms of focal adhesion kinase (FAK) were transiently transfected into MCF-7 cells using liposome reagents. CD98 stimulation increased the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV. Activation of CD98 augmented the invasion rate of MCF-7 cells through ECM. EDTA or a function-blocking anti-beta1 integrin mAb suppressed the effect of CD98 on invasiveness. Inhibition of phosphorylation of FAK by PP2, an inhibitor of Src family kinase, reduced CD98-induced invasion of MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, cytochalasin D or phalloidin inhibited CD98-mediated induction of tumor cell invasion. Inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated invasion of MCF-7 cells were diminished by pretreatment of cells with Mn++, which is shown to induce conformational change of beta1 intgerin. These results provide the first evidence that CD98 activation increases tumor cell invasion by activating beta1 integrin affinity, and that FAK phosphorylation and subsequent cytoskeletal reorganization may be essential for CD98-mediated regulation of cell motility.
Actins ; Antigens, CD29 ; Breast Neoplasms* ; Breast* ; Cell Movement ; Collagen ; Cytochalasin D ; Cytoskeleton ; Edetic Acid ; Extracellular Matrix ; Fibronectins ; Focal Adhesion Protein-Tyrosine Kinases ; Glycoproteins ; Humans* ; Immunohistochemistry ; Indicators and Reagents ; Laminin ; Liposomes ; MCF-7 Cells* ; Phalloidine ; Phosphorylation ; Phosphotransferases

OBJECTIVES: ICAM-1, VCAM-1, and betal integrins mediate cell-cell or cell-matrix interactions. We investigated effects of mixed leukocyte reaction (MLR), cyclosporine A (CsA), or hydrocortisone (HC) on their expression by endothelial (EnC) and mesangial cells (MC). METHODS: MLR was performed with or without CsA or HC for 5 days. After adding 25N MLR supernatant, cytokines or CsA to MC or EnC, the expression of VCAM-1, ICAM-1, and alpha3beta1 and a501 integrins was examined by using cell surface enzyme immunoassays or flow cytometry. RESULTS: MLR supernatant induced a marked increase in the expression of ICAM-1 and VCAM-1 on MC or EnC(p<0.001). HC treatment during MLR effectively inhibited MLR-induced upregulation of VCAM-1 and ICAM-1 on both cells (p<0.005). HC had, however, no inhibitory effect on VCAM-1 expression when added with MLR supernatant to cells. CsA treatment during MLR caused a modest decrease in MLR-induced expression of VCAM-1 on EnC, but had no effect on that of ICAM-1. INFgamma or TGFbeta1 stimulated expression of VCAM-1, and INFgamma, IL-1beta, or TNF alpha expression of ICAM-1 on MC after 24 hr. INFgamma, or TGFbeta1 enhanced expression of alpha3beta1 or alpha5beta1 integrins on MC after 5 days. CsA caused a modest decrease in basal expression of VCAM-1, and also decreased the basal, or INFgamma, or TGFbeta1-induced expression of alpha3beta1 and alpha5beta1 integrins on MC. CONCLUSION: Alloreactive lymphocytes and monocytes upregulate expression of VCAM-1 and ICAM-1 on EnC and MC maybe by secretion of cytokines such as INFgamma, and facilitate leukocytes attachment and following renal or vascular injury. HC effectively prevent the upregulation of VCAM-1 and ICAM-1 by inhibiting the release of cytokines during MLR. CsA did not cause an increase in the expression of VCAM-1 and beta1 integrin.
Antigens, CD29 ; Cyclosporine* ; Cytokines ; Flow Cytometry ; Hydrocortisone* ; Immunoenzyme Techniques ; Integrins ; Intercellular Adhesion Molecule-1 ; Leukocytes ; Lymphocyte Culture Test, Mixed* ; Lymphocytes ; Mesangial Cells* ; Monocytes ; Up-Regulation ; Vascular Cell Adhesion Molecule-1 ; Vascular System Injuries