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PubMed2003

THY1 expression is associated with tumor suppression of human ovarian cancer.

Microcell-mediated transfer of chromosome 11 into the human ovarian cancer cell line SKOV-3 results in suppression of tumorigenicity in severe combined immunodeficiency (SCID) mice. To identify the differentially expressed transcripts associated with suppression of tumorigenicity, cDNA populations from the slow-growing tumorigenic clone 11(H)8-3, tumorigenic clone 11(H)8-4, and parental SKOV-3 cells were subtracted from the nontumorigenic clones, 11(H)7-2 and 11(C)9-8. The subtracted cDNA populations were either cloned, sequenced and searched in GenBank, or analyzed by gene discovery array screening. A cDNA transcript corresponding to the THY1 gene located at chromosome 11q23 approximately q24 was found to be exclusively expressed in the two nontumorigenic cell clones. In contrast, THY1 expression was not detected in SKOV-3, the tumorigenic hybrid clones, or six other tumorigenic ovarian cancer cell lines. Further analysis using immunocytochemistry and quantitative flow cytometry with a Thy-1-specific antibody confirmed the exclusive expression of THY1 at the protein level in the two nontumorigenic clones. Several cell growth and differentiation-related genes, including thrombospondin 1 (THBS1), SPARC [secreted protein, acidic, cysteine-rich (osteonectin)], and fibronectin (FN1) were also found to be upregulated in the nontumorigenic clones; however, these were expressed in the slow-growing tumorigenic clones as well. Expression of these genes was not observed in the parental SKOV-3 cell line and therefore must be regulated by a gene or genes on chromosome 11. Our results suggest that THY1 is a putative tumor suppressor gene for ovarian cancer and that THBS1, SPARC, and FN1 are genes associated with the regulation of in vivo tumor growth rate.

Cell Division↗
PubMed2001

BM-40 and MMP-2 expression are not coregulated in human melanoma cell lines.

As published recently, suppression of BM-40 (osteonectin, SPARC) expression reduced the in vitro adhesive and invasive capacities of melanoma cells. In parallel, a decreased production of matrix metalloproteinase-2 (MMP-2) was observed in these cells indicating a close coregulation of these two proteins. However, analysis of 12 independently established melanoma cell lines, exhibiting different metastatic capacities, did not reveal a positive correlation between BM-40 or MMP-2 expression and the metastatic capacities of these cell lines. In addition, direct comparison of BM-40 and MMP-2 mRNA levels did not provide evidence for coregulation of these two proteins. This was further corroborated by the finding that exogenous BM-40 failed to alter synthesis and activation of proMMP-2.

Collagenases↗
PubMed2000

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Biomarkers↗
PubMed2003

Origin-associated features of chondrocytes in mouse Meckel's cartilage and costal cartilage: an in vitro study.

Using a cell culture method, we histochemically and immunohistochemically investigated whether chondrocytes deriving from different origins, such as Meckel's or costal cartilages, express similar phenotypic characteristics. Chondrocytes isolated enzymatically from Meckel's and costal cartilages of 17-day embryonic mice both actively proliferated and formed cartilage nodules consisting of toluidine blue-positive proteoglycans and type II collagen. Both deposited calcified cartilaginous matrix as revealed by alkaline phosphatase (ALPase) activity and alizarin red staining throughout 3 weeks in culture. Immunostaining for osteopontin (OP), osteocalcin (OC), and osteonectin (ON) revealed that chondrocytes from both cartilages were positive for their proteins, but type I collagen was detected only in cells transforming from Meckel's chondrocytes late in the culture. Electron microscopy demonstrated that although costal and Meckel's chondrocytes had typical chondrocytic features during 2 weeks in culture, Meckel's chondrocytes transformed into osteocytic cells that produced thick, banded type I collagen fibrils. In contrast, costal chondrocytes maintained typical hypertrophic morphology throughout the final stage of culture. The present study suggests that Meckel's chondrocytes derived from neural crest-ectomesenchyme retain osteogenic potential, and differ from costal chondrocytes originating from mesoderm.

Alkaline Phosphatase↗
PubMed1995

A purine-rich sequence in the human BM-40 gene promoter region is a prerequisite for maximum transcription.

BM-40 (osteonectin, SPARC [secreted protein, acidic, rich in cysteine]) is a highly conserved, matrix-associated protein that is found in basement membranes, bones and remodeling tissues throughout vertebrate evolution. We are reporting the characterization of the 5' end of the human BM-40 gene. Sequence comparison of the 5' region revealed significant homologies with the bovine and murine genes, including a purine-rich stretch composed of two boxes, GGA-box 1 and 2, separated by a pyrimidine-rich spacer element. Transfection analyses of the human BM-40 promoter provide strong evidence that this region comprises several distinct regulatory domains, to which different functions can be assigned. GGA-box 1 is thereby absolutely required and sufficient by itself for maximal BM-40 transcriptional activity, whereas the spacer element has a down-regulatory effect. Comparative transfection analyses in human cell lines, positive or negative for BM-40 transcripts, indicate that the GGA-box sequences in the human promoter, in contrast to the bovine promoter, do not significantly contribute to cell-type specific expression in human cells.

Adenosine↗
PubMed2002

UVB-induced premature senescence of human diploid skin fibroblasts.

In this work, we show that repeated stresses with UVB (290-320 nm) induce stress-induced premature senescence (SIPS) of skin human diploid fibroblasts (HDFs). HDFs at early cumulative population doublings were exposed three or five times to increasing subcytotoxic doses of UVB with one stress per day. After 2 days of recovery, several biomarkers of replicative senescence were established. First, there was an increase in the proportion of cells positive for senescence-associated beta-galactosidase activity. Second, there was a loss of replicative potential as assessed by a very low level of [3H]-thymidine incorporation. Third, the steady-state level of the mRNA of three senescence-associated genes, i.e. fibronectin, osteonectin and SM22, was increased in HDFs at 72 h after three and five exposures to UVB. In conclusion, these results suggest that it is possible to induce SIPS in HDFs after repeated exposures to subcytotoxic doses of UVB. This model could be used to test whether HDFs in UVB-induced premature senescence are able to promote epithelial cell growth and tumorigenesis in skin, as shown recently with HDFs in H(2)O(2)-induced premature senescence.

Cell Line↗
PubMed1996

Human osteosarcoma (OST) induces mouse reactive bone formation in xenograft system.

To distinguish the origin of bone-forming cells in the osteosarcoma (OST) tumor inoculated into nude mice, we have developed a novel in situ hybridization technique. The system used digoxygenin (DIG) labeled DNA probes that encoded human specific repetitive gene, Alu, and mouse specific repetitive gene, mouse L1 (m-L1). The chondrogenic and osteogenic cells in the tumor had strongly positive signals for m-L1 probe without any signals for Alu probe. The expression of bone matrix proteins was also examined by in situ hybridization. The bone-forming cells were positive for mRNAs of mouse osteonectin, osteopontin, and osteocalcin relating to calcification during bone formation, while these were negative for human mRNAs of these bone matrix proteins. The OST cells in the tumor expressed the human bone morphogenetic proteins (BMPs) mRNAs by RT-PCR. These data indicated that the mouse cells, not the human sarcoma cells, are responsible for cartilage and bone formation in the OST tumor inoculated into nude mice, and we speculated that BMPs, at least in part, could play an important role in this ossification.

Animals↗
PubMed1998

An isolation and in vitro culturing method for human intraoral bone cells derived from dental implant preparation sites.

In dental implantology, the biocompatibility of the osseous tissue to the implant surface and to local environmental factors plays an important role in the process of healing. Bone cells derived from intraoral osseous tissue proves to be an important source of the osteoprogenitor cells required for healing of the periodontium around implants. Historically, the rat calvaria model has been employed to study the effects of various dental treatments on bone in vitro. However, there are morphological and functional differences which exist between bone cells derived from rat calvaria and human intraoral osseous tissue that impose certain limitations on the usefulness of the rat calvaria model for dental implant applications. Therefore, an in vitro culturing method for the isolation, growth and maintenance of human intraoral bone cell cultures derived from osseous tissues is truly warranted. In addition, a method for the accurate characterization of these bone cells as osteoblasts is also vital. The specific objective of this study was to establish isolation and in vitro culturing methods utilizing human intraoral bone cells derived from dental implant preparation sites. This paper describes techniques for the harvesting of human bone cells from the intraoral derived osseous tissues and discuss the procedures for maintaining the primary intraoral bone cell culture. In addition, our studies utilize established protocols for the characterization of these cells as osteoblasts by means of alkaline phosphatase activity determination, identification of cellular osteonectin and osteocalcin antigens, establishing the presence of cells expressing type I collagen and determining the ability of cells to produce calcifications. The utilization of intraoral osseous tissue may prove useful for future dental implant research by providing an in vitro model system more closely related to conditions encountered clinically.

Adult↗
PubMed2000

Three-dimensional cellular development is essential for ex vivo formation of human bone.

Tissue engineering of human bone is a complex process, as the functional development of bone cells requires that regulatory signals be temporally and spatially ordered. The role of three-dimensional cellular interactions is well understood in embryonic osteogenesis, but in vitro correlates are lacking. Here we report that in vitro serum-free transforming growth factor (TGF)-beta1 stimulation of osteogenic cells immediately after passage results in the formation of three-dimensional cellular condensations (bone cell spheroids) within 24 to 48 hours. In turn, bone cell spheroid formation results in the up-regulation of several bone-related proteins (e.g., alkaline phosphatase, type I collagen, osteonectin) during days 3-7, and the concomitant formation of micro-crystalline bone. This system of ex vivo bone formation should provide important information on the physiological, biological and molecular basis of osteogenesis.

Alkaline Phosphatase↗
PubMed1996

Structure of a novel extracellular Ca(2+)-binding module in BM-40.

The EF-hand is a highly conserved Ca(2+)-binding motif found in many cytosolic Ca(2+)-modulated proteins. Here we report the crystal structure at 2.0 A resolution of the carboxy-terminal domain of human BM-40 (SPARC, osteonectin), an extracellular matrix protein containing an EF-hand pair. The two EF-hands interact canonically but their detailed structures are unusual. In the first EF-hand a one-residue insertion is accommodated by a cis-peptide bond and by substituting a carboxylate by a peptide carbonyl as a Ca2+ ligand. The second EF-hand is stabilized by a disulphide bond. The EF-hand pair interacts tightly with an amphiphilic amino-terminal helix, reminiscent of target peptide binding by calmodulin. The present structure defines a novel protein module occurring in several other extracellular proteins.

Amino Acid Sequence↗
PubMed2004

Overexpression of SPARC gene in human gastric carcinoma and its clinic-pathologic significance.

Gastric cancer is the second most common cancer in the world and the fifth leading cause of cancer-related death in Taiwan. To improve the survival of gastric cancer patients, biomarkers for early detection and effective anticancer therapy are required. An essential first step is to profile gene expression in gastric cancer and identify genes that are aberrantly expressed, and to do this cDNA microarrays were performed. The clinic-pathologic correlation and prognostic significance of the aberrantly expressed genes were evaluated to identify novel biomarkers of gastric cancer. Fresh surgical samples of tumour tissue and matching noncancerous mucosa were obtained immediately after gastric resection in 43 patients. Secreted Protein, Acidic and Rich in Cysteine (SPARC) (Osteonectins), one of the most highly expressed genes in both intestinal and diffuse gastric cancers in our microarray results, was selected for further study. The overexpression of SPARC was verified using real-time quantitative-reverse transcription-polymerase chain reaction (Q-RT-PCR), Northern blot and immunohistochemical staining. The expression of SPARC in tumour tissues was, on average, 4.27-fold increased (95% CI 2.68-5.85) compared to adjacent noncancerous mucosa (P<0.001). The expression of SPARC was higher in advanced (T2, T3 and T4) cancer compared to the early (T1) cancer (P=0.048) with regard to depth of wall invasion. Higher expression of SPARC was significantly associated with lymph node metastasis (P<0.001), lymphatic invasion (P=0.004) and perineural invasion (P=0.047). Expression of SPARC in patients in stage II and above was significantly higher than those in stage I (P=0.017). The 3-year survival of patients with lower expression of SPARC was significantly better than those with a higher expression (log rank P=0.047). These data indicate the potential of SPARC as a prognostic marker for gastric cancer.

Adult↗
PubMed2004

Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells.

Osteopontin (OPN), bone sialoprotein (BSPII), and osteonectin (ON) belong to a family of glycoproteins, which have been linked to cancer metastasis and progression. Here, we report on the selection of antisense oligonucleotides (ASOs), which are effective in reducing their protein levels. In human MDA-MB-231 breast cancer cells, the maximum inhibition of protein expression ranged from 84% (OPN) to 75% (BSPII) and 70% (ON). Erucylphospho-NNN-trimethylpropanolamine (ErPC3) was used as positive control and combination partner. Exposure to ErPC3 inhibited colony formation of MDA-MB-231 cells by 11% (10 microM), 45% (14 microM) and 78% (20 microM). The clonogenicity of breast cancer cells was reduced by 15%, 11%, 8% (5 microM), 39%, 19%, 14% (10 microM) and 46%, 39%, 21% (20 microM) in response to ASO-OPN-04, ASO-BSPII-06 and ASO-ON-03, respectively. Combination of ErPC3 with the ASOs caused additive combination effects. Pre-exposure to the ASOs, but not to the NSO, inhibited formation of osteolytic metastasis in three of four (ASO-OPN-04, P<0.03) and two of four (ASO-BSPII-06) nude rats, and reduced metastasis lesions significantly (T/C%=4.3 and 9.1, P=0.05, respectively). We conclude that downregulation of OPN and BSPII reduces colony formation of MDA-MB-231 cells and formation of osteolytic metastasis in nude rats.

Animals↗
PubMed2000

Down-regulation of the extracellular matrix protein SPARC in vSrc- and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo.

In vitro transformation of primary cultures of chick embryo fibroblasts by the membrane-bound vSrc or the nuclear vJun oncoproteins is correlated with a down-regulation of the secreted glycoprotein SPARC (also called BM-40 or osteonectin). This protein is a nonstructural component of the extracellular matrix that is thought to regulate cell-matrix interaction during development, wound repair, and carcinogenesis. Its precise function remains unclear. To estimate the contribution of SPARC down-regulation to the major aspects of the transformed phenotype, we have reexpressed this protein from a self-replicating retrovirus Rcas, designated R-SPARC, in the transformed cultures. These R-SPARC-infected cultures display the following main properties: (i) they accumulate the SPARC protein to a level identical to or only slightly higher than the level in normal chick embryo fibroblasts; (ii) they retain the main phenotypic properties characteristic of in vitro transformed cells, that is, altered morphology, capacity to grow in a reduced amount of serum, and capacity to develop colonies from single cells in agar; (iii) they display a clearly reduced capacity to develop local fibrosarcomas in vivo. Taken together, these data strongly suggest that down-regulation of SPARC contributes to the transformed phenotype triggered by vSrc and vJun in primary avian fibroblasts, by facilitating in vivo tumorigenesis.

Animals↗
PubMed1998

Studies of multimerin in patients with von Willebrand disease and platelet von Willebrand factor deficiency.

In normal platelet alpha-granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron-lucent zone. Because the platelet stores of VWF are deficient in 'platelet low' type 1 and type 3 von Willebrand disease (VWD), we investigated their electron-lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal alpha-granular multimerin and factor V, and normal alpha-granular fibrinogen, thrombospondin-1, fibronectin, osteonectin and P-selectin. In type 3 VWD platelets, alpha-granular electron-lucent zones lacking VWF-associated tubules were identified and multimerin was found in its normal alpha-granular location. These findings indicate that the formation of the electron-lucent zone and the sorting of multimerin to this region occur independent of VWE The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of 'platelet low' type 1 VWD.

Blood Platelets↗
PubMed2000

Expression of osteopontin messenger RNA by macrophages in ovarian serous papillary cystadenocarcinoma: a possible association with calcification of psammoma bodies.

Calcified psammoma bodies often appear in human ovarian serous papillary cystadenocarcinoma. Osteocalcin (OC), osteonectin (ON) and osteopontin (OPN) are three members of non-collagenous bone-related proteins known to be related with mineralization of bone. To clarify possible involvement of these bone matrix proteins in the calcification of the psammoma bodies, the expression of OC, ON and OPN was analyzed by immunohistochemical and in situ hybridization studies using 15 surgical specimens. OPN protein was detected in the calcified area of the psammoma bodies which was positively stained by von Kóssa's staining, while OC and ON proteins were not. OPN protein was not detected in any cells in tissues, but OPN messenger ribonucleic acid (mRNA) was detected in CD68-positive macrophages, indicating that OPN was produced and promptly secreted by macrophages. These results suggest that OPN produced and promptly secreted by macrophages and subsequently translocated to psammoma bodies may be causally related with the calcium phosphate deposition in the psammoma bodies of the ovarian serous papillary cystadenocarcinomas.

Antigens, CD↗
PubMed2001

SPARC expression in primary human renal cell carcinoma: upregulation of SPARC in sarcomatoid renal carcinoma.

SPARC (secreted protein acidic and rich in cysteine, also called osteonectin, BM-40, and 43K protein) is a matricellular protein and is associated with cell-matrix interactions during cell proliferation and extracellular remodeling. It is also implicated in the neovascularization, invasion, and metastasis of human malignancies. To investigate a potential role of the SPARC in renal tumorigenesis, we examined primary renal cell carcinomas (RCCs) for SPARC expression by Northern blot analysis and for protein distribution by immunohistochemistry. We found that 6 (100%) of 6 sarcomatoid and 25 (70%) of 36 clear-cell carcinomas had enhanced SPARC transcription compared with that of the corresponding normal kidney tissue. In contrast, papillary and chromophobe RCCs characterized by a hypovascular or avascular tumor phenotype had undetectable SPARC expression. Immunohistochemical analysis showed that SPARC was strongly stained in the cytoplasm of the sarcomatoid neoplastic cells in sarcomatoid RCCs, whereas it was expressed only in the vascular endothelial cells and fibroblasts in clear-cell RCCs. SPARC staining intensity in the stromal cells was increased in the invading portion in some clear-cell RCCs. These findings suggest that tumor development, including neovascularization and invasion in clear-cell RCCs, might be regulated by SPARC from stromal endothelial cells and fibroblasts and that sarcomatoid transformation from common-type RCCs is associated with upregulation of SPARC expression; SPARC may contribute to its aggressive tumor phenotype.

Blotting, Northern↗
PubMed2000

Cell differentiation and bone protein synthesis in the lungs of sheep with spontaneous calcinosis.

The lungs of three sheep with spontaneous enzootic calcinosis induced by the calcinogenic plant Nierembergia veitchii (Nv) were examined electron microscopically and immunohistochemically. The main ultrastructural changes were activation of fibroblasts and modified smooth muscle cells (SMCs) in the pulmonary interstitium, with an increase in extracellular matrix and precipitation of calcium, either in a laminated pattern or as amorphous aggregates. Macrophages and multinucleated giant cells, some with calcium crystals in the cytoplasm, were found in areas of increased extracellular matrix. Thickening and replication of the basal lamina of capillaries were prominent. The bone proteins osteocalcin, osteopontin and osteonectin were detected immunohistochemically in the cytoplasm of activated fibroblasts, in modified SMCs and in the extracellular matrix. It is suggested that 1,25-dihydroxyvitamin D(3)in Nv induces cellular differentiation and the synthesis of a calcifiable matrix.

Animals↗
PubMed2000

Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins.

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.

Adenosine Triphosphate↗