Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.     

Mineralization process during acellular cementogenesis in rat molars: a histochemical and immunohistochemical study using fresh-frozen sections

This study was designed to detect tissue non-specific alkaline phosphatase (TNSALP) by Azo-dye staining, calcium by glyoxal bis (2-hydroxyanil) (GBHA) staining, bone sialoprotein (BSP) and osteopontin (OPN) by immunoperoxidase staining in developing rat molars, and also to discuss the mineralization process during acellular cementogenesis. To restrain a reduction in histochemical and immunohistochemical reactions, fresh-frozen undemineralized sections were prepared. Where the epithelial sheath was intact, TNSALP reaction was observed in the dental follicle, but not in the epithelial sheath. With the onset of dentin mineralization, the BSP- and OPN-immunoreactive, initial cementum layer appeared. At this point, cementoblasts had shown intense TNSALP reaction and GBHA reactive particles (=calcium-GBHA complex) appeared on the root surface. With further development, the reaction of TNSALP and GBHA became weak on the root surface. Previous studies have shown that the initial cementum is fibril-poor and that matrix vesicles and calciferous spherules appear on the root surface only during the initial cementogenesis. The findings mentioned above suggest that: during the initial cementogenesis, cementoblasts release matrix vesicles which result in calciferous spherules, corresponding to the GBHA reactive particles. The calciferous spherules trigger the mineralization of the initial cementum. After principal fiber attachment, mineralization advances along collagen fibrils without matrix vesicles.     

Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth

Experimental periodontal regeneration studies have revealed the weak binding of repair cementum to the root surface, whereas attachment of cementum to dentin preconditioned by odontoclasts appears to be superior. The aim of this study has been, therefore, to analyze the structural and partial biochemical nature of the interface that develops between resorbed dentin and repair cementum by using human deciduous teeth as a model. Aldehyde-fixed and decalcified tooth samples were embedded in acrylic or epoxy resins and sectioned for light and transmission electron microscopy. Antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two noncollagenous proteins accumulating at hard tissue interfaces in bone and teeth, were used for protein A-gold immunocytochemistry. Light microscopy revealed a gradually increasing staining intensity of the external dentin matrix starting after the withdrawal of the odontoclast. Labeling for both BSP and OPN was first detected among the exposed collagen fibrils and in the intratubular dentin matrix when odontoclasts had withdrawn but mesenchymal cells were present. Subsequently, collagen fibrils of the repair cementum were deposited concomitantly with the appearance of labeling for BSP and OPN over the intratubular, intertubular, and peritubular dentin matrix. Labeled mineralization foci indicated the advancing mineralization front, and the collagenous repair matrix became integrated in an electron-dense organic material that showed labeling for BSP and OPN. Thus, no distinct planar interfacial matrix layer lies between the resorbed dentin and the repair cementum. The results suggest that odontoclasts precondition the dentin matrix such that the repair cementum becomes firmly attached.This study was supported by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, University of Berne, Berne, Switzerland.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

Ultrastructural immunolocalization of dentin matrix protein 1 on Sharpey's fibers in monkey tooth cementum

Despite the importance of dentin matrix protein 1 (DMP1) in the formation of mineralized tissue, including dentinogenesis and osteogenesis, its precise role in cementogenesis remains to be clarified fully. The purpose of our study was to demonstrate the ultrastructural immunolocalization of DMP1 in monkey molar tooth cementum. Japanese Macaca fuscata monkeys were fixed by perfusion. The upper molar teeth and accompanying periodontium then were dissected and demineralized with EDTA. Cryosections were obtained, incubated in anti-DMP1 polyclonal antibody, and processed by immunoperoxidase and immunogold labeling. Intense immunoperoxidase staining for DMP1 was observed in acellular extrinsic fiber cementum, particularly in Sharpey's fibers. Cementocyte lacunae with canaliculi showed DMP1 staining in the apical region of the tooth root. Electron immunomicroscopy revealed the close proximity of DMP1 to collagen fibrils in Sharpey's fibers at the mineralization front. Intense immunogold labeling was localized on the walls of the cementocyte lacunae in cellular cementum. These results should contribute to better understanding of the role of DMP1, not only in Sharpey's fiber biomineralization, but also in the maintenance of the cementocyte lacunar space in cementum.     

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages

Background information. Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non‐collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages. Results. DFCs were isolated from tooth germs of SD (Sprague—Dawley) rats and then co‐cultured with dNCPs. Treated DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. The expression of mineralization‐associated proteins and genes were up‐regulated after induction, whereas the expression of specific markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealed that a large amount of cementum‐like tissues was formed within the novel dentin carriers, which were quite distinct from the newly formed osteodentin secreted by DPSCs (dental pulp stem cells). The negative expression of DSP (dentin sialoprotein) also excluded the possibility of producing dentin matrix by treated DFCs. Conclusions. dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.     

Early expression of bone matrix proteins in osteogenic cell cultures.

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.     

Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture

Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient's dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration.     

Immunohistochemical localization of calbindin D28k during root formation of rat molar teeth

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig's epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca(2+) concentration.     

The mosasaur tooth attachment apparatus as paradigm for the evolution of the gnathostome periodontium

SUMMARY Vertebrate teeth are attached to jaws by a variety of mechanisms, including acrodont, pleurodont, and thecodont modes of attachment. Recent studies have suggested that various modes of attachment exist within each subcategory. Especially squamates feature a broad diversity of modes of attachment. Here we have investigated tooth attachment tissues in the late cretaceous mosasaur Clidastes and compared mosasaur tooth attachment with modes of attachment found in other extant reptiles. Using histologic analysis of ultrathin ground sections, four distinct mineralized tissues that anchor mosasaur teeth to the jaw were identified: (i) an acellular cementum layer at the interface between root and cellular cementum, (ii) a massive cone consisting of trabecular cellular cementum, (iii) the mineralized periodontal ligament containing mineralized Sharpey's fibers, and (iv) the interdental ridges connecting adjacent teeth. The complex, multilayered attachment apparatus in mosasaurs was compared with attachment tissues in extant reptiles, including Iguana and Caiman . Based on our comparative analysis we postulate the presence of a quadruple-layer tissue architecture underlying reptilian tooth attachment, comprised of acellular cementum, cellular cementum, mineralized periodontal ligament, and interdental ridge (alveolar bone). We propose that the mineralization status of the periodontal ligament is a dynamic feature in vertebrate evolution subject to functional adaptation.     

Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.     

Effects of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) on the formation of dentin and the periodontal attachment apparatus in the mouse

The effects of the bisphosphonate HEBP on dentin formation and the structural relationship between the dentin and the developing periodontal attachment apparatus have been studied in the continuously growing mouse incisor. It was observed that HEBP (in doses greater than or equal to 8 mg P/kg b.w/day) not only inhibited the deposition of mineral crystallites in newly formed dentin matrix, but also entirely prevented the formation of a layer of acellular root cementum. It was further noticed that the drug interfered with the deposition of 3H-serine-containing substances at the predentin-dentin border. This was not always accompanied by an inhibition of dentin mineralization, thereby suggesting that 3H-serine-containing proteins (presumably phosphoproteins) do not play a critical role in the deposition of mineral layers onto previously formed ones. The absence of a cementum layer did not prevent the developing periodontal ligament from establishing matrix-to-matrix connections with the root-analogue dentin. Collagen fibrils of the ligament intermingled with those of the mantle dentin, which in contrast to teeth not exposed to the drug were clearly visible and not masked by electron-dense matrix components. Finally, it was found that the drug had distinct effects on the formation of root-analogue and crown-analogue dentin. Whereas during the course of the experiment the odontoblasts along the crown-analogue aspect of the tooth continued to produce circumpulpal dentin matrix, those along the root-analogue aspect of the tooth did so only when the mantle dentin layer had been mineralized prior to HEBP administration. This phenomenon is interpreted as being indicative of fundamental differences between the formation of crown and root dentin.     

Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.     

Synthesis and processing of bone sialoproteins during de novo bone formation in vitro.

Bone sialoprotein (BSP) and osteopontin (OPN) are sulphated and phosphorylated sialoglycoproteins that regulate the formation of hydroxyapatite crystals during de novo bone formation. To gain insights into the relationship between the synthesis and posttranslational modification of BSP and OPN and the mineralization of bone, pulse-chase studies were conducted on cultures of newly forming bone nodules produced by fetal rat calvarial cells in vitro. Cultures were pulse labelled with 35SO4, or with either 32PO4 or [gamma-32P]ATP to study intracellular and extracellular phosphorylation, respectively, and chased in isotope-free medium for various times up to 24 h. The presence of radiolabelled BSP and OPN was determined in the cells, in culture medium, and in various tissue compartments obtained by dissociative extraction with 4 M GuHCl (G1), 0.5 M EDTA (E), and again with 4 M GuHCl (G2) and a bacterial collagenase digestion of the demineralized collagenous tissue residue. With each isotope employed, radiolabelled BSP and OPN were detected in the E extract within the 1-h chase period and increased in amount with time. Similarly, 35SO4- and 32PO4-labelled BSP increased in the G2 extract, but OPN was not detected. In the G1 extract the 35SO4-labelled BSP decreased with chase time, whereas the 32PO4-labelled BSP increased. No differences were evident in the profiles of BSP labelled with 32PO4 or [gamma-32P]ATP. In the absence of beta-glycerophosphate, which is required for optimal mineralization of the bone nodules, 35SO4-labelled BSP was increased in the medium and G1 extract and decreased in the E extract and G2 extract after 3 h. In addition to differences in the tissue compartmentalization of BSP and OPN, these studies indicate that 35SO4 is lost from BSP during mineralization and that isoforms of BSP exist with a selective affinity for the organic and mineral phases. Moreover, the additional phosphorylation of BSP and OPN catalyzed by ectokinase activity does not appear to alter the distribution of these sialoproteins.     

Establishment of acellular extrinsic fiber cementum on human teeth

Summary The present study describes for the first time the development of early acellular extrinsic fiber cementum (AEFC) until its establishment on human teeth. Precisely selected premolars with roots developed to 50%–100% of their final length were prefixed in Karnovsky's fixative and most of them were decalcified in EDTA. Their roots were subdivided into about 10 blocks each, cut from the mesial and distal root surfaces. Following osmication, these blocks were embedded in Epon and sectioned for light-and transmission electron microscopy. Some blocks were cut non-demineralized. From semithin stained sections, the density of the collagenous fiber fringe protruding from the root surface was measured by using the Videoplan-system. After initiation of this fiber fringe and its attachment to the dentinal root surface followed by mineralization, the fringe gradually increased in length and subsequently became mineralized. Fringe elongation and the advancement of the mineralization front appeared to progress proportionally. Thus, in all stages of AEFC development, a short fiber fringe covered the mineralized AEFC. Its density remained constant, irrespective of AEFC thickness. The latter gradually increased and reached an early maximum of 15–20 m in the cervical region. At this stage, the AEFC fringe appeared to fuse with the future dentogingival or other collagen fibers of the tooth supporting apparatus. Mineralization of the fringe commenced with isolated, spherical or globular centers, which later fused with the mineralization front and became incorporated in AEFC.Abbreviations AEFC acellular extrinsic fiber cementum - CIFC cellular intrinsic fiber cementum - CMSC cellular mixed stratified cementum - CEJ cemento-enamel junction - CM centers of mineralization - D dentin - DCJ dentino-cemental junction - EDTA ethylene diaminetetraacetic acid - FF fiber fringe - GL glycogen storage granules - MF mineralization front - PL periodontal ligament - PLF periodontal ligament fibers     

Formation of intermediate cementum. II: a scanning electron microscopic study of the epithelial root sheath of Hertwig in monkey

The intermediate cementum is a layer of calcified tissue between the dentin and the cementum at the periphery of dental roots. The mineralization pattern of the intermediate cementum and the innermost layer of aprismatic enamel in the crowns has been shown to be very similar. Since the formation of these tissues is incompletely known and there have been diverging opinions whether they are of epithelial or mesenchymal origin, the present study aimed at investigating the surface morphology of the root sheath with scanning electron microscopy and correlate it to the mineralization of the intermediate cementum. The advancing mineralization front of the intermediate cementum was covered by the root sheath. Numerous microvilli facing the root were found on this part of the root sheath. The corresponding surface facing the dental follicle was covered with bulb-type junctions. Microvilli and bulb-type junctions have also been demonstrated on the surface of the presecretory ameloblast and associated with formation of aprismatic enamel. Thus, the epithelial root sheath seems to actively take part in the formation of intermediate cementum.     

Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack

Metastatic cancer cells, like trophoblasts of the developing placenta, are invasive and must escape immune surveillance to survive. Complement has long been thought to play a significant role in the tumor surveillance mechanism. Bone sialoprotein (BSP) and osteopontin (OPN, ETA-1) are expressed by trophoblasts and are strongly up-regulated by many tumors. Indeed, BSP has been shown to be a positive indicator of the invasive potential of some tumors. In this report, we show that BSP and OPN form rapid and tight complexes with complement Factor H. Besides its key role in regulating complement-mediated cell lysis, Factor H also appears to play a role when "hijacked" by invading organisms in enabling cellular evasion of complement. We have investigated whether BSP and OPN may play a similar role in tumor cell complement evasion by testing to see whether these glycoproteins could promote tumor cell survival. Recombinant OPN and BSP can protect murine erythroleukemia cells from attack by human complement as well as human MCF-7 breast cancer cells and U-266 myeloma cells from attack by guinea pig complement. The mechanism of this gain of function by tumor cell expression of BSP or OPN has been defined using specific peptides and antibodies to block BSP and OPN protective activity. The expression of BSP and OPN in tumor cells provides a selective advantage for survival via initial binding to alpha(V)beta(3) integrin (both) or CD44 (OPN) on the cell surface, followed by sequestration of Factor H to the cell surface and inhibition of complement-mediated cell lysis.