Tumor-derived Osteopontin Is Soluble, Not Matrix Associated
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m109028200
ISSN1083-351X
AutoresSusan R. Rittling, Yanping Chen, Fei Feng, Yiming Wu,
Tópico(s)dental development and anomalies
ResumoThe secreted phosphoprotein osteopontin (OPN), when immobilized on a surface, supports cell adhesion, prevents apoptosis of endothelial cells, and is a ligand for the αvβ3 integrin, which is important in endothelial cell biology and neovascularization. OPN synthesized by tumor cells stimulates tumor growth, but the mechanism by which the protein acts remains unclear. One possibility, therefore, is that OPN may exert its effects on tumor growth by enhancing angiogenesis. While OPN is found at high levels in bone, where it is a component of the mineralized matrix, we have asked here whether OPN present in tumors is similarly extracellular matrix associated. We have shown that OPN is detectable in tumor extracts and in serum of tumor-bearing mice, and that the protein in tumors and in serum can be synthesized by both tumor and the host cells. Biochemical fractionation of tumor tissue confirmed that there is little if any association of OPN with the insoluble fraction. Immunochemical analysis of murine mammary tumors shows no co-localization of OPN with the extracellular matrix, identified by laminin staining. Ras-transformed cells in culture produce abundant OPN, however, the protein was found to be associated with the cell fraction but not with the matrix fraction. An enzyme-linked immunosorbent assay was used to demonstrate that OPN in conditioned medium from these cells fails to associate with extracellular matrix components, including laminin and fibronectin,in vitro. Recombinant OPN (GST-OPN) when coated onto a plastic surface can support human umbilical vein endothelial cell adhesion, suppressing apoptosis and allowing cell cycle progression, at concentrations from 1 to 50 μg/ml. Soluble GST-OPN in the same concentration range has no effect on HUVECs held in suspension. Thus, we conclude that OPN associated with tumors is primarily soluble, and that soluble OPN can neither support endothelial cell proliferation nor prevent apoptosis of these cells in the absence of adhesion. The secreted phosphoprotein osteopontin (OPN), when immobilized on a surface, supports cell adhesion, prevents apoptosis of endothelial cells, and is a ligand for the αvβ3 integrin, which is important in endothelial cell biology and neovascularization. OPN synthesized by tumor cells stimulates tumor growth, but the mechanism by which the protein acts remains unclear. One possibility, therefore, is that OPN may exert its effects on tumor growth by enhancing angiogenesis. While OPN is found at high levels in bone, where it is a component of the mineralized matrix, we have asked here whether OPN present in tumors is similarly extracellular matrix associated. We have shown that OPN is detectable in tumor extracts and in serum of tumor-bearing mice, and that the protein in tumors and in serum can be synthesized by both tumor and the host cells. Biochemical fractionation of tumor tissue confirmed that there is little if any association of OPN with the insoluble fraction. Immunochemical analysis of murine mammary tumors shows no co-localization of OPN with the extracellular matrix, identified by laminin staining. Ras-transformed cells in culture produce abundant OPN, however, the protein was found to be associated with the cell fraction but not with the matrix fraction. An enzyme-linked immunosorbent assay was used to demonstrate that OPN in conditioned medium from these cells fails to associate with extracellular matrix components, including laminin and fibronectin,in vitro. Recombinant OPN (GST-OPN) when coated onto a plastic surface can support human umbilical vein endothelial cell adhesion, suppressing apoptosis and allowing cell cycle progression, at concentrations from 1 to 50 μg/ml. Soluble GST-OPN in the same concentration range has no effect on HUVECs held in suspension. Thus, we conclude that OPN associated with tumors is primarily soluble, and that soluble OPN can neither support endothelial cell proliferation nor prevent apoptosis of these cells in the absence of adhesion. osteopontin fibronectin BSA, bovine serum albumin phosphate-buffered saline extracellular matrix human umbilical vein endothelial cells fibroblast growth factor population doubling level insulin, transferrin, selenium 2,2′-azinobis(3-benzathiazoline-6-sulfonic acid) enzyme-linked immunosorbent assay The secreted phosphoprotein osteopontin is synthesized by both osteoblasts and osteoclasts, and accumulates in bone (1Butler W.T. Ridall A.L. McKee M.D. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles in Bone Biology. Academic Press, San Diego1996: 167-181Google Scholar), where it is required for optimal osteoclast function (2Yoshitake H. Rittling S.R. Denhardt D.T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8156-8160Crossref PubMed Scopus (318) Google Scholar). OPN1 is also expressed in a variety of other tissues, notably those with a major epithelial component, where it is excreted into a variety of fluids: the function of the protein in these tissues is less well defined. The protein is expressed at high levels in a variety of tumors and transformed cells (3Rittling S.R. Novick K.W. Cell Growth Differ. 1997; 8: 1061-1069PubMed Google Scholar, 4Denhardt D.T. Giachelli C. Rittling S.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 723-749Crossref PubMed Scopus (308) Google Scholar, 5Casson A.G. Wilson S.M. McCart J.A. O'Malley F.P. Ozcelik H. Tsao M.S. Chambers A.F. Int. J. Cancer. 1997; 72: 739-745Crossref PubMed Scopus (72) Google Scholar, 6Brown L.F. 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The function of OPN in tumorigenesis is still not clear. In particular, the cell type(s) with which OPN interacts to stimulate tumor growth is not known. One well described function of OPN in vitro is as a cell adhesion molecule when it is coated onto a plastic surface (9Chambers A.F. Hota C. Prince C.W. Cancer Res. 1993; 53: 701-706PubMed Google Scholar, 10Senger D.R. Perruzzi C.A. Papadopoulous-Sergiou A. Van De Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (182) Google Scholar). The protein contains an RGD sequence that can mediate cell adhesion (11Bautista D.S. Xuan J.W. Hota C. Chambers A.F. Harris J.F. J. Biol. Chem. 1994; 269: 23280-23285Abstract Full Text PDF PubMed Google Scholar), although other non-RGD sequences in the molecule can also mediate adhesion: these include the SVVYGLR sequence in human OPN (12Yokosaki Y. Matsuura N. Sasaki T. Murakami I. Schneider H. Higashiyama S. Saitoh Y. Yamakido M. Taooka Y. Sheppard D. J. Biol. 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Adhesion of cells to OPN is in most cases through a variety of integrins (reviewed in Ref. 18Denhardt D.T. Noda M. J. Cell. Biochem. 1998; S30/31: 92-102Crossref Google Scholar, see also Refs. 13Barry S.T. Ludbrook S.B. Murrison E. Horgan C.M. Biochem. Biophys. Res. Commun. 2000; 267: 764-769Crossref PubMed Scopus (75) Google Scholar and 19Caltabiano S. Hum W.T. Attwell G.J. Gralnick D.N. Budman L.J. Cannistraci A.M. Bex F.J. Biochem. Pharmacol. 1999; 58: 1567-1578Crossref PubMed Scopus (23) Google Scholar); the non-integrin receptor CD44 may also mediate cell adhesion to OPN (15Katagiri Y.U. Sleeman J. Fujii H. Herrlich P. Hotta H. Tanaka K. Chikuma S. Yagita H. Okumura K. Murakami M. Saiki I. Chambers A.F. Uede T. Cancer Res. 1999; 59: 219-226PubMed Google Scholar,20Weber G.F. Ashkar S. Glimcher M.J. Cantor H. Science. 1996; 271: 509-512Crossref PubMed Scopus (820) Google Scholar). Most notably, OPN is a well characterized ligand for the αvβ3 integrin, which is important for angiogenesis and endothelial cell survival (21Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2185) Google Scholar, 22Eliceiri B.P. Cheresh D.A. J. Clin. Invest. 1999; 103: 1227-1230Crossref PubMed Scopus (619) Google Scholar). Thus, one possibility for the function of OPN in tumorigenesis is to promote survival of endothelial cells: indeed the protein has been demonstrated to have this function in vitro, as an insoluble adhesive ligand (23Scatena M. Almeida M. Chaisson M.L. Fausto N. Nicosia R.F. Giachelli C.M. J. Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (448) Google Scholar). Like endothelial cells, osteoclasts express high levels of the αvβ3 integrin: this integrin is critical for osteoclast function (24McHugh K.P. Hodivala-Dilke K. Zheng M.H. Namba N. Lam J. Novack D. Feng X. Ross F.P. Hynes R.O. Teitelbaum S.L. J. Clin. Invest. 2000; 105: 433-440Crossref PubMed Scopus (592) Google Scholar). OPN is present at high levels in bone (25McKee M.D. Nanci A. Microsc. Res. Tech. 1996; 33: 141-164Crossref PubMed Scopus (306) Google Scholar, 26Prince C.W. Oosawa T. Butler W.T. Tomana M. Bhown A.S. Bhown M. Schrohenloher R.E. J. Biol. Chem. 1987; 262: 2900-2907Abstract Full Text PDF PubMed Google Scholar), and is an important ligand for the osteoclast αvβ3 integrin as evidenced by reduced bone resorption in osteopontin-deficient mice (2Yoshitake H. Rittling S.R. Denhardt D.T. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8156-8160Crossref PubMed Scopus (318) Google Scholar). Thus, in bone, OPN is a well established component of the extracellular matrix, and can function in vivo in this tissue as a cell attachment molecule. However, osteopontin has a high affinity for hydroxyapatite by virtue of its general acidic nature, its high degree of phosphorylation, as well as due to a run of 9–10 aspartates in the NH2-terminal half of the molecule (27Reinholt F.P. Hultenby K. Oldberg A. Heinegard D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4473-4475Crossref PubMed Scopus (705) Google Scholar). Thus, its accumulation in bone may be due to its affinity for the mineral, rather than the protein component of bone tissue. It is still not clear, therefore, if OPN functions as a cell attachment molecule in soft tissues. While numerous cell types have been demonstrated to adhere to OPN coated onto a plastic surface, including endothelial cells, fibroblasts, transformed cells, and smooth muscle cells (10Senger D.R. Perruzzi C.A. Papadopoulous-Sergiou A. Van De Water L. Mol. Biol. Cell. 1994; 5: 565-574Crossref PubMed Scopus (182) Google Scholar, 11Bautista D.S. Xuan J.W. Hota C. Chambers A.F. Harris J.F. J. Biol. Chem. 1994; 269: 23280-23285Abstract Full Text PDF PubMed Google Scholar, 23Scatena M. Almeida M. Chaisson M.L. Fausto N. Nicosia R.F. Giachelli C.M. J. Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (448) Google Scholar, 28Liaw L. Almeida M. Hart C.E. Schwartz S.M. Giachelli C.M. Circ. Res. 1994; 74: 214-224Crossref PubMed Scopus (375) Google Scholar, 29Somerman M.J. Prince C.W. Sauk J.J. Foster R.A. Butler W.T. Matrix. 1987; 9: 49-54Crossref Scopus (80) Google Scholar, 30Zheng D.Q. Woodard A.S. Tallini G. Languino L.R. J. Biol. Chem. 2000; 275: 24565-24574Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), this activity is only physiologically relevant if these cell types encounter immobilized OPN in vivo. An important question to be addressed therefore is whether OPN is associated with the extracellular matrix in non-calcified matrices. Of special interest is the situation in tumors: if OPN, which is expressed at quite high levels in many tumor types, is functioning as a cell adhesion molecule it should be immobilized in the matrix. In the work presented here, we have used a variety of techniques to localize OPN in tumor tissues and cells, and conclude that OPN made by tumors is primarily, if not exclusively, soluble. Laminin (Sigma), fibronectin (Invitrogen), collagen I and matrigel (Collaborative Research/BD) were obtained from commercial sources, while recombinant GST-OPN was isolated as described (31Xuan J.W. Hota C. Chambers A.F. J. Cell. Biochem. 1994; 54: 247-255Crossref PubMed Scopus (74) Google Scholar). mOPN was isolated from ras-transformed 3T3 cell conditioned medium by using DEAE-Sepharose chromatography. Antibodies to laminin and fibronectin were obtained from Sigma. Antibodies to OPN were: goat anti-rat OPN antibody OP199 (28Liaw L. Almeida M. Hart C.E. Schwartz S.M. Giachelli C.M. Circ. Res. 1994; 74: 214-224Crossref PubMed Scopus (375) Google Scholar) or rabbit anti-human OPN antibody LF 123 (32Fisher L.W. Stubbs J.T.I. Young M.F. Acta Orthop. Scand. 1995; 66: 61-65Crossref Scopus (0) Google Scholar): both these antibodies were used at a dilution of 1:1000; they both react with mouse OPN and were used interchangeably. LF123 has a slightly higher affinity for mouse OPN, but cross-reacts nonspecifically with a few other bands in tumor and cell lysates: the specificity of these antibodies has been described in detail elsewhere (33Rittling S.R. Feng F. Biochem. Biophys. Res. Commun. 1998; 250: 287-292Crossref PubMed Scopus (42) Google Scholar). For immunohistochemistry, pooled, affinity purified mouse anti-mouse OPN 2A. J. Kowalski, S. R. Rittling, and D. T. Denhardt, unpublished data. was used. For immunofluorescence, monoclonal anti-mouse 2A12 was used; this antibody reacts with an epitope in the C-terminal half of OPN. These antibodies were raised in the OPN −/− mice. The tumors used here were derived from two different experimental systems. Mammary tumors arising spontaneously in MMTV-c-myc/MMTV-v-Ha-rastransgenic mice, either wild type or OPN-deficient (34Feng F. Rittling S.R. Breast Cancer Res. Treat. 2000; 63: 71-79Crossref PubMed Scopus (25) Google Scholar), were used for analysis of extracellular matrix. For the analysis of OPN accumulation in sera and tumors in hosts of different genotypes, tumors were induced by injection of ras-transformed 3T3 cells as described (8Wu Y.M. Denhardt D.T. Rittling S.R. Br. J. Cancer. 2000; 83: 156-163Crossref PubMed Scopus (80) Google Scholar) into either wild type or OPN-deficient mice. In all cases, mice were sacrificed by exsangination, and tumors were removed and immediately flash frozen on liquid nitrogen. Powdered frozen tissue was stored at −70 °C until use. Tumor extracts were made by resuspending the powdered tissue in RIPA buffer containing 0.1 mmphenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml aprotinin, and subjecting the extract to 3 cycles of freeze/thaw. For preparation of extracellular matrix from mammary tumors, 50 mg of powdered tissue were resuspended in 50 mm Tris, pH 7.5, with 0.2% Triton X-100 containing protease inhibitors as above (Tris/Triton), and sonicated. The suspension was centrifuged (15,000 × g; 5 min) and the supernatant retained. The pellet was resuspended in Tris/Triton, and subjected to two more cycles of sonication and centrifugation. The final pellet was resuspended in 50 mm Tris, pH 7.5, 6 m urea. OPN in serum was collected by addition of 1/10 volume each of 15% barium chloride and 3.8% sodium citrate on ice. The resulting precipitate was collected, and washed at 0 °C sequentially with 0.5 volume of 15% barium chloride and H2O. Proteins were eluted by boiling the washed pellets in 20 μl of SDS loading buffer containing 0.2m sodium citrate. Western blotting was performed as described (33Rittling S.R. Feng F. Biochem. Biophys. Res. Commun. 1998; 250: 287-292Crossref PubMed Scopus (42) Google Scholar). ras-transformed wild type and OPN-deficient 3T3 cell lines (8Wu Y.M. Denhardt D.T. Rittling S.R. Br. J. Cancer. 2000; 83: 156-163Crossref PubMed Scopus (80) Google Scholar) were plated in Dulbecco's modified Eagle's medium containing 3% fetal bovine serum on 35-mm wells previously coated with 0.3 mg/ml collagen I in 20 mm acetic acid or with 0.7 mg/ml matrigel (Collaborative Research). The cells were allowed to become confluent and the extracellular matrix and soluble fractions obtained using a modification of the procedure of Gospodarowicz and co-workers (35Globus R.K. Plouet J. Gospodarowicz D. Endocrinology. 1989; 124: 1539-1547Crossref PubMed Scopus (268) Google Scholar). Briefly, the cells were washed and lysed in 10 mmTris, pH 7.5, 0.5% Triton X-100, 10 mmphenylmethylsulfonyl fluoride, and the supernatant retained. The wells were then washed with 20 mm NH4OH in 10 mm Tris, pH 7.5. Material remaining in the well was scraped into 10 mm Tris, pH 7.5, containing 2% SDS. Control wells (whole cell extracts) were solubilized directly in 2% SDS. Alternatively, the cells were removed with 3 mm EDTA in PBS, after which the matrix was washed and solubilized as above. These methods are commonly used for the preparation of extracellular matrix (36Burgess J.W. Gould D.R. Marcel Y.L. J. Biol. Chem. 1998; 273: 5645-5654Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 37Owensby D.A. Morton P.A. Schwartz A.L. J. Biol. Chem. 1989; 264: 18180-18187Abstract Full Text PDF PubMed Google Scholar). An alternative method, removal of cells by incubation with cytochalasin B, was not effective for these transformed cells (data not shown). For OPN and fibronectin, 1/5 of the cell or ECM extracts were separated by PAGE; for laminin, 1/60 of the extracts were used. Equal proportions rather than equal amounts of protein were used because the cell lysate contains proportionately much more protein than the ECM. Wells of 96-well plates were coated with matrix components at 200 μg/well in PBS overnight at 4 °C. These wells were blocked the next day with 2% BSA. Conditioned medium from either wild type or OPN-deficient ras-transformed cells was then added to the coated well and incubated at room temperature for 2 h. The same conditioned medium, and dilutions as indicated were applied directly to uncoated wells. All wells were then washed with water, and the matrix containing wells, and some of the wells containing directly coated conditioned medium were fixed with 4% paraformaldehyde for 30 min at room temperature. Following additional washes, OPN was detected with affinity purified polyclonal antimouse OPN2 at 1:3000 dilution, followed by horseradish peroxidase-labeled goat anti-mouse IgG (Bio-Rad) at a 1:1000 dilution. After washing the horseradish peroxidase substrate, ABTS (0.3 mg/ml in 55 mm citrate, pH 4.0, with .03% H2O2) was added and incubated for 10–30 min in the dark. Serial sections frommyc/ras mammary tumors (38Pattengale P.K. Stewart T.A. Leder A. Sinn E. Tepler I. Schmidt E. Leder P. Am. J. Pathol. 1989; 135: 39-61PubMed Google Scholar) (fixed in methacarn and embedded in paraffin) were rehydrated and endogenous peroxidases blocked in 3% H2O2 in methanol. Tumors arising in both wild type and OPN −/− mice were used (34Feng F. Rittling S.R. Breast Cancer Res. Treat. 2000; 63: 71-79Crossref PubMed Scopus (25) Google Scholar). Sections were blocked with goat serum (1:20 in PBS) for 30 min. Primary antibodies were diluted in 1% BSA, 1% goat serum, and incubated with tissue sections for 1 h or overnight. Affinity purified polyclonal anti-mouse OPN (1:1000) was biotinylated with the DAKO ARK system before application to the tissue. Rabbit anti-laminin was from Sigma, was used at 1:300 and detected with biotinylated goat anti-rabbit IgG. All sections were reacted with the ABC reagent (Vector ABC Elite) prior to visualization of the antibody reactivity by staining with diaminobenzidine (Sigma Fast DAB). Sections were lightly counterstained with hematoxylin and mounted in permount. Control sections were incubated without primary antibody. Paraformaldehyde sections frommyc/ras mammary tumors (38Pattengale P.K. Stewart T.A. Leder A. Sinn E. Tepler I. Schmidt E. Leder P. Am. J. Pathol. 1989; 135: 39-61PubMed Google Scholar) were rehydrated and subjected to antigen retrieval (0.01 m citrate, pH 6.0, 100 °C, 10 min). Sections were blocked with goat serum (1:20 in PBS) for 1–2 h. Primary antibodies were diluted in 1% BSA, 1% goat serum, and incubated with tissue sections overnight. Affinity purified monoclonal antibody 2A1 at 0.18 μg/ml was biotinylated with the DAKO ARK system before application to the tissue, and was detected with rhodamine-avidin D (Vector Labs, 16 μg/ml). Anti-laminin was detected with fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Jackson). Rhodamine and fluorescein isothiocyanate signals were collected and a single optical section is shown. Controls with no primary antibody showed fluorescence that was indistinguishable from untreated tissue (data not shown). Human umbilical vein endothelial cells (HUVECs; Clonetics from pooled donors) were cultured in M199 with 10% fetal bovine serum (Hyclone), 4 ng/ml aFGF, 75 μg/ml endothelial cell growth supplement (Sigma), and 5 units/ml heparin, and used before PDL 15. Prior to experiments, confluent cells were incubated overnight in M199 with 1 × ITS supplement (Invitrogen), in the absence of serum and growth factors. Quiescent cells were detached with trypsin, washed with M199, and plated at 5 × 104cells/well in M199 containing 1 × ITS and 2% BSA in 48-well plates coated with different substrates. Wells were coated as described below. For apoptosis, cells were incubated overnight in the absence of growth factors, and then labeled for 15 min with 8 μg/ml Hoescht 33342. Floating and adherent cells were collected, fixed, and counted under fluorescence illumination. For [3H]thymidine incorporation, cells were incubated overnight with growth factors (10 ng/ml FGF plus 5 units/ml heparin), then 2 μCi/ml [3H]thymidine were added and the cells were incubated for an additional 5–6 h. Cells were washed, fixed in 7% trichloroacetic acid, then solubilized in 0.5 n NaOH, 0.5% SDS and counted. Control experiments showed that this protocol recovered all the trichloroacetic acid-precipitable counts in the wells. For adhesion assays, exponentially growing cells in complete medium were harvested with trypsin, resuspended in complete medium, and washed in Dulbecco's modified Eagle's medium. Cells were resuspended in 2% BSA, and plated in 96-well plates (Immulon II) at 2 × 104 cells/well. The wells had been previously coated with different substrates as indicated. After 2 h, the wells were washed and the cells stained with crystal violet. Following elution of the dye with acetic acid, absorbance of the wells at 630 nm was determined. Substrates were diluted in PBS and coated on the wells overnight at 4 °C; the wells were subsequently blocked with 2% BSA for 30 min at 37 °C. Substrates used were mOPN (OPN purified fromras-transformed 3T3 cells), GST-OPN (recombinant mouse OPN as a GST fusion protein), fibronectin (Invitrogen), laminin (Sigma), and recombinant GST protein. Unless otherwise indicated, proteins were used at 10 μg/ml. ras-transformed 3T3 lines were generated from both wild type and OPN-deficient mice: these cell lines have been previously described (8Wu Y.M. Denhardt D.T. Rittling S.R. Br. J. Cancer. 2000; 83: 156-163Crossref PubMed Scopus (80) Google Scholar), and shown to give rise to tumors following subcutaneous injection into syngeneic wild type hosts. These tumors form much more slowly when the injected cells are deficient for OPN production, illustrating the important role that OPN plays in the process of tumorigenesis (8Wu Y.M. Denhardt D.T. Rittling S.R. Br. J. Cancer. 2000; 83: 156-163Crossref PubMed Scopus (80) Google Scholar). In other experiments (data not shown), these same cell lines were injected into control (wild type) and OPN-deficient host mice, and tumors allowed to develop. When the tumors reached a volume of 2000–3000 mm3, serum was prepared from the tumor-bearing animals and the tumors were excised. OPN levels in these sera and in tumor extracts were analyzed by Western blotting as shown in Fig. 1. When wild type, OPN-expressing tumors developed in wild type or in OPN-deficient hosts (Fig. 1 A, lanes 2–4 and9–11), OPN protein accumulated to significant levels in the serum. This is consistent with the high level of expression of OPN in the wild type cell lines. Interestingly, in wild type animals bearing OPN-deficient tumors, serum levels of OPN were again elevated over control (non-tumor bearing, wild type mice) levels (Fig. 1 A,lanes 5–7). OPN was not detected in the serum of OPN-deficient mice bearing OPN-deficient tumors (data not shown). Extracts of OPN-expressing tumors similarly contained OPN, as shown previously for OPN-expressing tumors developed in wild type hosts (3Rittling S.R. Novick K.W. Cell Growth Differ. 1997; 8: 1061-1069PubMed Google Scholar,34Feng F. Rittling S.R. Breast Cancer Res. Treat. 2000; 63: 71-79Crossref PubMed Scopus (25) Google Scholar), and the amount of protein was similar whether or not the host animal expressed OPN (Fig. 1 B, lanes 1–2 and 5–6). OPN-deficient tumors developed in wild type mice expressed easily detectable OPN, and in some cases the level of OPN in these tumors was as high as that in wild type tumors (Fig. 1 B,lanes 3 and 4). Again, no OPN was detected when extracts from OPN-deficient tumors developed in OPN-deficient hosts were analyzed (Fig. 1 B, lane 7). We conclude from these results that much of the OPN made by tumors is secreted into the circulation, and that sufficient OPN is produced by the host cells in response to tumor growth to result in substantial levels of the protein in the tumor itself (as detected by Western blotting), and also in elevated serum levels. Biochemical fractionation of tumor extracts was performed to determine whether the OPN present in tumors was associated with the ECM. Tumors arising spontaneously in transgenic mice expressing myc and v-Ha-ras specifically in the mammary gland (34Feng F. Rittling S.R. Breast Cancer Res. Treat. 2000; 63: 71-79Crossref PubMed Scopus (25) Google Scholar, 38Pattengale P.K. Stewart T.A. Leder A. Sinn E. Tepler I. Schmidt E. Leder P. Am. J. Pathol. 1989; 135: 39-61PubMed Google Scholar) were flash-frozen and resuspended in Triton X-100 containing buffer, sonicated to disrupt the cell structure, and insoluble material collected by centrifugation. Following three cycles of resuspension and sonication in the same buffer, equal amounts of protein from the first supernatant and the final pellet were analyzed by SDS-PAGE followed by Western blotting (Fig. 2). The extracellular matrix proteins fibronectin and laminin were retained in the pellet fraction, confirming that these pellets indeed contained the extracellular matrix material. OPN, on the other hand, was localized exclusively in the soluble fraction: no OPN immunoreactivity could be detected in the pellet, at least in the amount of protein loaded in the well of the PAGE gel (30 μg). Cross-reacting proteins identified in both the soluble and pellet fractions are present in both WT and OPN −/− tumors, indicating that they are not OPN. Laminin and fibronectin reactivity appear also in the soluble fraction: this reactivity may be due to soluble protein present in the tumor, or to partial solubilization of the ECM by the Triton/sonication protocol used. Even if the latter is the case, there is a clear differentiation between the pattern of association of OPN and the former proteins with the insoluble pellet. The localization of osteopontin in tumor tissue was further examined in these spontaneously arising tumors by immunohistochemistry. For these experiments we chose to use the spontaneously arising tumors rather than those resulting from subcutaneous injection of transformed cells, reasoning that the spontaneous tumors would have a more extensive and well developed extracellular matrix. Accumulation of OPN was variable in these tumors, with areas of high expression and regions where OPN was undetectable (Fig. 3: panel B, OPN +/+). Some tumor samples expressed very low levels of OPN (data not shown). In regions where OPN expression was high, however, immunoreactivity appeared over tumor cell cytoplasm. The extracellular matrix in these tumors was identified by staining of adjacent sections for laminin, which was associated particularly with connective tissues and with the basement membrane of blood vessels (Fig. 3: panels A and D, LAM), with some reactivity appearing associated with the tumor cells themselves. Double immunofluorescence for OPN and laminin, examined at higher magnification in a single optical section by confocal
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