Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D3 on differentiation of a human osteosarcoma (MG-63).

Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.     

Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.     

Bone matrix osteonectin limits prostate cancer cell growth and survival

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.     

Three-dimensional type I collagen co-culture systems for the study of cell-cell interactions and treatment response in bone metastases

The available monolayer culture systems for the study of bone metastases constitute a suboptimal simulation of the in vivo pathophysiology of bone metastases, and therefore, do not provide sufficient information to assess the morphologic evidence of bone reaction to cancer cells, the nature of cell-specific mediators of osteolysis and osteoplasia and the response to treatment. Therefore, we have developed a three-dimensional (3-D) type I collagen gel system that allows co-culture of human osteoblasts (MG-63) with cancer cells, such as MCF-7, MDA-MB-231 or ZR-75 breast cancer cells, PC-3 prostate cancer, KLE endometrial cancer cells and Calu-1 lung cancer cells. We used type I collagen purified from rat tail tendons and the 3-D system was prepared by mixing MG-63 cells with type I collagen in 24-well plates. The 3-D system was inoculated with cancer cells and processed with standard cell culture procedures. After 1 week of culture, the matrix gel was fixed with formalin and embedded in paraffin. Serial sections were stained with trichrome Masson stain and modified Masson-Goldner stain, as well as analyzed by in situ hybridization, immunohistochemistry and the TUNEL technique for semi-quantitative detection of apoptotic cell death, assessing the response to adriamycin therapy. The inoculation of PC-3 cells in this collagen matrix produced a blastic reaction, documented by an increased number of MG-63 cells and increased density of type I collagen. The human KLE cells and inoculation of cell-free media produced no reaction, while ZR-75, MCF-7 and Calu-1 cells produced local degradation of the collagen matrix. In situ hybridization revealed the expression of Insulin-like growth factor 1 (IGF-1) and urokinase-type plasminogen activator (uPA) mRNA, while immunohistochemistry detected differential expression of uPA and cathepsin D. Adriamycin induced apoptotic cell death in prostate cancer cells and estrogen receptor negative (ER-) MDA-MB-231 breast cancer cells, while adriamycin did not induce apoptosis but cytostasis in ER+ MCF-7 cells. The adriamycin-induced apoptosis was inhibited by co-culture with osteoblast-like cells (MG-63). We conclude that this 3-D culture system is a useful in vitro model allowing the analysis of local mediators of osteolytic and osteoblastic reactions to bone metastases and treatment response.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.     

Establishment of an osseous cell line from fetal rat calvaria using an immunocytolytic method of cell selection: characterization of the cell line and of derived clones

Using selective media and complement-mediated lysis of primary cultures of a fetal rat calvarial cell population, we have developed a cell line (OBCK6) that exhibits osteoblastic characteristics. OBCK6 cells demonstrated enhanced parathyroid hormone (PTH)-stimulated adenylate cyclase activity relative to the primary calvarial population, production of alkaline phosphatase activity and type 1 collagen, and the capacity to form mineralized nodules in unsupplemented medium after prolonged (22-26 day) culture. Two sublines, CFK1 and CFK2, which were isolated by dilution cloning, differed morphologically and with respect to growth rate. CFK1 cells demonstrated high PTH and prostaglandin E2-stimulated adenylate cyclase activity, whereas only low PTH-stimulated activity was observed in CFK2 cells. Retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] each reduced PTH-stimulated adenylate cyclase activity in both the cell types. Retinoic acid and dexamethasone reduced and 1,25(OH)2D3 enhanced alkaline phosphatase activity in these cells. PTH significantly augmented alkaline phosphatase activity to a much greater extent in CFK1 than in CFK2 cells. Both CFK1 and CFK2 cells expressed type I but type III collagen, and neither expressed osteocalcin. Strong Alcian blue staining of CFK2 cells was suggestive of a cartilaginous phenotype. These three cell lines, therefore, demonstrated discrete characteristics of skeletal cell function and should provide important models for evaluation of mechanisms of mineralization and for control of skeletal cell growth and mesenchymal differentiation in vitro.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3

Background  

Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.Key words: dental pulp stem cell, mesenchymal stem cells, osteogenic differentiation, 3D scaffolds.     

Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative.

Collagen has been reported to be essential for the proliferation of various kinds of cells including human osteoblastic cells [Takamizawa, S., Maehata, Y., Imai, K., Senoo, H., Sato, S., Hata, R., 2004. Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol. Int. 28, 255-265], but the type(s) of collagen responsible for growth regulation is not known. Presently we found that ascorbic acid 2-phosphate, a long-acting vitamin C derivative, stimulated both cell growth and the expression of mRNA for type III collagen in human osteoblast-like MG-63 cells and in normal human osteoblasts, as well as in human bone marrow mesenchymal stem cells, but not the expression of type I collagen in these cells. Epidermal growth factor also stimulated both cell growth and expression of type III collagen mRNA in MG-63 cells. Among MG-63 cell clones, their growth rates correlated significantly with their COL3A1 messenger RNA levels but not with their COL1A1 or COL1A2 messenger RNA levels. Transfection of MG-63 cells with siRNA for COL3A1 but not with that for COL1A1 decreased the growth rates of the transfected cells concomitant with a drop in the level of COL3A1 mRNA. Furthermore, cell proliferation as observed by thymidine incorporation into DNA and cell number was increased when MG-63 cells were cultured on type III collagen-coated dishes. Taken together, our results indicate that type III collagen is the collagen component responsible for the growth stimulation of human osteoblastic cells.     

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.     

An Epstein-Barr virus-transformed B cell line produces autoregulatory interleukin-1 that regulates bone remodeling

In this study, we demonstrate that an Epstein-Barr virus-transformed B cell line, A-11, produced interleukin-1 (IL-1), a cytokine that regulates bone remodeling. A-11 cells produce IL-1 in a cell dose- and culture time-related manner. The IL-1 activity was neutralized by recombinant human IL-1 (rhIL-1) alpha antiserum, but not by rhIL-1 beta antiserum. The IL-1 was semi-purified by (NH4)2SO4 precipitation, Superose prep 12 gel filtration, and anion-exchange chromatography strongly stimulated in vitro bone resorption. The stimulatory effect of the purified IL-1 on bone resorption was prostaglandin independent. Purified IL-1 inhibited DNA and collagen synthesis in the osteoblastic cell line MC3T3-E1. However, it enhanced significantly the cellular activity of alkaline phosphatase (EC 3.1.3.1), a marker enzyme for differentiation of osteoblasts. On the other hand, A-11 cell proliferation was inhibited by addition of rhIL-1 alpha antiserum, but not by rhIL-1 beta antiserum. And cell proliferation was stimulated by exogenous rhIL-1 alpha and -beta.     

Establishment of a Novel In Vitro Test Setup for Electric and Magnetic Stimulation of Human Osteoblasts

When large defects occur, bone regeneration can be supported by bone grafting and biophysical stimuli like electric and magnetic stimulation (EMS). Clinically established EMS modes are external coils and surgical implants like an electroinductive screw system, which combines a magnetic and electric field, e.g., for the treatment of avascular bone necrosis or pseudarthrosis. For optimization of this implant system, an in vitro test setup was designed to investigate effects of EMS on human osteoblasts on different 3D scaffolds (based on calcium phosphate and collagen). Prior to the cell experiments, numerical simulations of the setup, as well as experimental validation, via measurements of the electric parameters induced by EMS were conducted. Human osteoblasts (3 × 10⁵ cells) were seeded onto the scaffolds and cultivated. After 24 h, screw implants (Stryker ASNIS III s-series) were centered in the scaffolds, and EMS was applied (3 × 45 min per day at 20 Hz) for 3 days. Cell viability and collagen type 1 (Col1) synthesis were determined subsequently. Numerical simulation and validation showed an adequate distribution of the electric field within the scaffolds. Experimental measurements of the electric potential revealed only minimal deviation from the simulation. Cell response to stimulation varied with scaffold material and mode of stimulation. EMS-stimulated cells exhibited a significant decrease of metabolic activity in particular on collagen scaffolds. In contrast, the Col1/metabolic activity ratio was significantly increased on collagen and non-sintered calcium phosphate scaffolds after 3 days. Exclusive magnetic stimulation showed similar but nonsignificant tendencies in metabolic activity and Col1 synthesis. The cell tests demonstrate that the new test setup is a valuable tool for in vitro testing and parameter optimization of the clinically used electroinductive screw system. It combines magnetic and electric stimulation, allowing in vitro investigations of its influence on human osteoblasts.     

Inhibition of cell adhesion by type V collagen.

Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its alpha 1 chain, but not by its alpha 2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c 3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the alpha 1 chain of heat-denatured type V collagen.     

Osteomimicry of Mammary Adenocarcinoma Cells In Vitro; Increased Expression of Bone Matrix Proteins and Proliferation within a 3D Collagen Environment

Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.