PI3-K/Akt-mediated anoikis resistance of human osteosarcoma cells requires Src activation.

Considerable advances in understanding the mechanisms associated with anoikis resistance of normal and malignant epithelial cells have been made. However, little is still known about the pathways involved in anoikis resistance of non-epithelial cells such as fibroblasts and sarcomas. Our results show that Src activity contributes to anoikis resistance of human osteosarcoma SAOS-2 cells. Src was found to be upregulated in anoikis resistant SAOS cells, and pharmacological inhibition of its activity resulted in the restoration of anoikis sensitivity. A normal pattern of dephosphorylation of FAK was observed upon cell detachment of both anoikis sensitive and resistant SAOS-2 cells, suggesting that FAK activity during anoikis resistance is not essential. The activity of Akt was found to be upregulated in anoikis resistant SAOSar cells and the pharmacological inhibition of PI3-K activity restored sensitivity to anoikis resistant cells, reconfirming the critical role of PI3-K/Akt pathway in cell survival. Furthermore, pharmacological inhibition of Src resulted in a decrease of Akt phosphorylation at Ser473. Altogether, these studies indicated a survival pathway mediated by the Src-dependent activation of the PI3-K/Akt pathway in a manner independent of FAK activity.

A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment.

Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser74, located in the Ser74-Gly75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH2-terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser74-Gly75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.

Colocalization of dentin matrix protein 1 and dentin sialoprotein at late stages of rat molar development.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.

Detection of dentin sialoprotein in rat periodontium.

Cloning and sequencing of the cDNA indicates that dentin sialophosphoprotein (DSPP) is a precursor of both dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Dentin sialophosphoprotein must be proteolytically processed to form these two extracellular matrix (ECM) proteins. Numerous studies led us to conclude that DSP (and DSPP) are exclusively expressed by odontoblasts and preameloblasts. However, recent observations suggest a wider distribution. To test this hypothesis, we conducted systematic studies on rat first molar during root formation with immunohistochemical techniques using specific anti-DSP polyclonal and monoclonal antibodies. We also performed in situ hybridization, using high-stringency RNA probes to detect DSP transcripts. Immunohistochemical studies demonstrated that DSP is not only localized in odontoblasts, dentin ECM and preameloblasts, but also in alveolar bone, cellular cementum, osteocytes, cementocytes, and their matrices. The results of in situ hybridization were consistent with those from immunohistochemistry, showing the expression of DSP transcripts in osteoblasts of alveolar bone, fibroblasts in periodontal ligament and cementoblasts in cellular cementum. Together, these observations suggest that DSP is involved in formation of the periodontium as well as tooth structures.

Dentin sialoprotein isoforms: detection and characterization of a high molecular weight dentin sialoprotein.

Dentin sialoprotein (DSP) is a glycoprotein accounting for 5-8% of the dentin non-collagenous proteins. The cDNA sequence predicts that rat DSP has 13 potential casein kinase phosphorylation sites and six potential N-linked glycosylation sites. However, its total phosphorylation level, as well as the nature and locations of the carbohydrate moieties, are unknown. Our findings in the present study show that rat DSP has 6.2 phosphates per molecule and that the majority of carbohydrates are attached to the protein through N-linked glycosylations. During our separation of dentin non-collagenous proteins with ion-exchange chromatography, we observed high molecular weight components eluting late in the salt gradient that were recognized by anti-DSP antibodies. We have purified these high molecular weight components using a monoclonal anti-DSP antibody affinity column. Data from amino acid analysis, phosphate level measurements and Edman degradation of tryptic peptides unequivocally proved that the very acidic, high molecular weight components are isoforms of DSP (designated HMW-DSP). Deglycosylation analysis indicates that the slower migration rate of HMW-DSP on SDS-PAGE results from its higher level of carbohydrate modifications.

Alpha 4 integrin increases anoikis of human osteosarcoma cells.

Cell motility, growth, and proliferation are regulated by adhesion to the extracellular matrix. Detachment of adherent cells from extracellular matrix results in induction of apoptosis ("anoikis"). Transformed cells often show an anchorage-independent growth that enables them to acquire a motile, invasive phenotype. This phenotype has been associated with the altered expression and function of the integrin family of transmembrane proteins that mediate cell adhesion to the extracellular matrix. Although alpha4 integrin is normally expressed on leukocyte subpopulations, a number of metastatic melanomas and sarcomas express it as well. In this study, we demonstrated the expression of alpha4 integrins on the human osteosarcoma cell line SAOS and on metastatic osteosarcoma lesions from the lung and pericardium. We further demonstrated that alpha4 integrin is coupled to the beta1 subunit by biochemical analysis and by using a mAb directed against a combinatorial epitope unique to the alpha4beta1 molecule. SAOS cells undergo anoikis when adherence is denied. Anoikis involved the activation of caspase 3 and the release of cytochrome c from mitochondria. Treatment of non-adherent SAOS with an anti-alpha4 mAb increased anoikis while anti-beta1 integrin mAbs did not alter anoikis, thus indicating a novel function for the alpha4 subunit in the control of cell death. Since integrins can control cell migration, proliferation, and apoptosis these results demonstrate a potential role for alpha4 integrin during multiple aspects of osteosarcoma metastasis.