Carsten Haeckel

Expression of cathepsin K in the human embryo and fetus.

Cathepsin K is a protease with high collagenolytic and elastinolytic activity. Its cellular expression was previously thought to be restricted to osteoclasts and osteoclast‐mediated bone resorption. In this study, the expression of cathepsin K in the human embryo and fetus was demonstrated by immunohistochemistry, in situ hybridization, and by Northern blotting of fetal tissue extracts. Besides osteoclasts and chondroclasts and their precursors, epithelial cells of various organ systems expressed significant amounts of this enzyme. Respiratory and gastrointestinal mucosa, including bile duct epithelia and urothelia, showed high levels of cathepsin K expression. With the exception of the urothelium, showing a more homogenous expression pattern, the protease was usually accentuated in the surface cell layers of pithelia. In summary, these findings in the human embryo and early fetus demonstrated a significant expression of cathepsin K in different epithelial cell types besides osteoclasts. The functional aspects of cathepsin K expression in nonosteoclastic cells and potential conclusions on physiological and pathological conditions in the embryo‐fetal or adult organism remain to be investigated. Dev Dyn 1999;216:89–95.

p53 Gene mutations in osteosarcomas of low-grade malignancy

Alterations in tumor suppressor gene p53, localized on chromosome 17p13, are considered to play a significant role in the initiation and, to some extent, even in the progression of various malignant tumors. In this respect, investigations on conventional highly malignant osteosarcomas have shown a mutation rate of approximately 20%. However, currently, data on the mutation rate in the group of variant histology osteosarcomas of low-grade malignancy do not exist. Therefore, we investigated a panel of low malignant entities (five low malignant intramedullary osteosarcomas grade 1; one intramedullary osteosarcoma grade 2; eight parosteal osteosarcomas, including one local recurrence grades 1 and 2, and five periosteal osteosarcomas grade 2) with polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis focusing on exons 4 to 8 of the p53 gene followed by direct sequencing. Point mutations were found in one low-grade osteoblastoma-like osteosarcoma and in two periosteal osteosarcomas grade 2 (one missense, one silent, and one nonsense mutation). This mutation rate of 15.7% (3 of 19) is comparable to that determined in highly malignant osteosarcomas. Moreover, the analysis of clinical data did not show any difference in the behavior of tumors with p53 mutations compared with those without. Therefore, we suggest that alterations in p53 gene are an early event in the tumorigenesis of malignant osteoblastic tumors without impact on progression of these tumors.

Prognostic significance of p53 gene mutations and p53 protein expression in synovial sarcomas.

Abstractu2002Alterations to p53 seem to be of prognostic significance in soft tissue sarcomas, but their significance for synovial sarcomas has not been studied. We analysed 34 synovial sarcomas in 19 patients for p53 alterations (p53 gene mutations + p53 immunopositivity) and examined this factor for its prognostic value in a group of 15 primary tumours. DNA was prepared from paraffin-embedded tumour material by a modified proteinase K/phenol/chloroform extraction. p53 gene mutations of exons 5–8 were analysed by the PCR-SSCP-sequencing method. p53 protein expression was evaluated by immunohistochemistry using the murine monoclonal antibody DO1. We found two missense mutations (5.9%) and ten p53 immunopositive cases (29.4%). Both tumours with p53 mutations showed p53 protein expression. There was no significant correlation between p53 alteration and histological subtype, age, sex, or tumour size. The 5-year survival rate was 24.1%. Overall survival was significantly reduced in patients having synovial sarcomas with p53 alterations (P<0.001). In the multivariate Cox’s analysis, only p53 alterations (P=0.032) and tumour size (P=0.023) emerged as independent prognostic factors. We suggest that p53 alterations may be a useful prognostic indicator in synovial sarcomas, allowing rational clinical treatment and follow-up.

Possible contribution of aminopeptidase N (APN/CD13) to invasive potential enhanced by interleukin-6 and soluble interleukin-6 receptor in human osteosarcoma cell lines.

This study aimed at clarifying the role of Aminopeptidase N (APN), a Zn2+-dependent ectopeptidase localized on the cell surface of human osteosarcoma cell lines treated with proinflammatory cytokines. We investigated the proinflammatory cytokines interleukin-1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α) as well as the anti-inflammatory cytokine transforming growth factor beta (TGF-β) for their influence on APN regulation. Soluble IL-6 receptor (sIL-6R) was always used together with IL-6 to achieve a stable effect. In addition, the invasive potential of the osteosarcoma cell lines MG63 and HOS was examined. Competitive RT-PCR and Ala-pNA activity assays revealed that IL-6 and sIL-6R significantly increased the mRNA expression and activity of APN in both osteosarcoma cell lines. Although IL-1β significantly stimulated APN mRNA expression in both cell lines, it influenced the enzyme activity only in MG63. TNF-α and TGF-β, however, had an effect neither on mRNA expression nor on the enzyme activity of APN in both cell lines. In the Matrigel invasion assay, IL-6 and sIL-6R significantly up-regulated the transmigration of these cell lines, whereas other cytokines did not. The up-regulated invasion was inhibited by bestatin, a specific inhibitor of APN. Cellular migration correlated highly with APN activity (r = 0.79, P < 0.002). These findings suggest that APN contributes to the invasive potential of human osteosarcomas enhanced by IL-6 and SIL-6R.

Inhibitory effect of antisense aminopeptidase N (APN/CD13) cDNA transfection on the invasive potential of osteosarcoma cells

AbstractAminopeptidase N (APN/CD13), a Zn2+-dependent ectopeptidase, is localized on the cell surface and functions as a transmembrane protein. Increased expression and activity of APN have been postulated to correlate with the aggressive behavior of several tumor types. In this study, the osteosarcoma cell line MNNG/HOS was stably transfected with an expression vector capable of expressing the antisense transcript of APN. Four stably transfected clones, the control clones and parental cells were characterized. Stable integration of the antisense vector was confirmed by PCR analysis of genomic DNA. Competitive RT-PCR revealed that mRNA expression of antisense-transfectants was decreased to approximately 37% of the control cell line. The activity assay showed that the enzymatic activity of APN was inhibited to approximately 51% of the control cell line. Antisense-transfection had no influence on the cellular proliferation measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, on the motility in Transwell chambers, and on the adhesive potential to collagen I. However, an in vitro invasion assay revealed a significant decrease in the number of cells that migrated through a reconstituted membrane (51% of the control cell line). The adhesive potential to Matrigel was also affected (73% of the control cell line). Furthermore, under in vivo conditions, a reduced potency to metastasize to the lung was shown in an experimental metastasis assay in nude mice. These findings demonstrate that APN plays an active role in the cellular attachment and proteolytic degradation of the extracellular matrix in the metastatic process of osteosarcomas.n Abbreviation: GAPDH – glyceraldehyde-3-phosphate dehydrogenase

Antisense inhibition of urokinase: effect on malignancy in a human osteosarcoma cell line.

Expression of urokinase‐type plasminogen activator (u‐PA) strongly correlates with a malignant tumor cell phenotype. In the multistep process of metastasis, different cellular functions are influenced by urokinase. The enzyme is known to be effective via both proteolytical and signal transduction mechanisms. In the present study, the osteosarcoma cell line MNNG/HOS was transfected with a vector capable of expressing an antisense transcript, complementary to 1,021 bases of the 3′ end of u‐PA cDNA. This construct was most effective in reducing u‐PA expression in previous experiments. Stably transfected antisense (as) cell lines were characterized and compared with the parental MNNG/HOS. Antisense transfection of MNNG/HOS gave the following results: (1) stable incorporation of the construct into the genome of as‐clones, as detected by Southern blot analysis; (2) decreased mRNA level of u‐PA, as detected by Northern blot analysis; (3) approximately 50% reduced enzyme expression in cell culture medium and cell homogenate; and (4) unchanged cellular proliferation activity and u‐PAR expression. In further functional analysis, as‐clones showed (1) significantly reduced invasion and motility in modified Transwell chambers (random migration and chemotaxis with collagen I as a chemoattractant); (2) significantly reduced adhesion on matrices of collagen I and vitronectin; (3) unchanged adhesion properties on Matrigel matrix; and (4) reduced metastatic potential to lungs and especially liver in chick embryos after i.v. infection into chorioallantoic membrane veins. Our data show that in MNNG/HOS urokinase influences cellular malignancy by promoting migration and selective adhesion. These specific functions were notable in addition to the effects on invasion and basement membrane degradation. Int. J. Cancer 77:153–160, 1998.© 1998 Wiley‐Liss, Inc.

Protease expression in dedifferentiated parosteal osteosarcoma

BACKGROUNDnParosteal osteosarcoma with dedifferentiation provides a useful model to study tumor progression from an indolent locally aggressive neoplasm to highly lethal metastasizing malignancy. Up-regulation of the proteolytic enzymes participating in stromal degradation is known to promote invasive growth and metastasis of several human and experimental tumors.nnnMETHODSnThe expression patterns of urokinasase plasminogen activator (u-PA), its cell-surface receptor (u-PAR), and cathepsin B were analyzed by immunohistochemical techniques in 11 cases of parosteal osteosarcoma and in 4 cases of dedifferentiated parosteal osteosarcoma.nnnRESULTSnBoth enzymes and the receptor were coexpressed in most tumor cells of parosteal and dedifferentiated parosteal osteosarcoma. Their expression was strikingly enhanced in the dedifferentiated high-grade component of the tumors. Tumor cells involved in bone production (ie, those adjacent to tumor produced bone trabeculae) exhibited equally strong expression of u-PA, u-PAR, and cathepsin B, regardless of their histologic grade. Expression of u-PA, u-PAR, and cathepsin B was undetectable in the normalized cells embedded in the well-developed tumor bone trabeculae.nnnCONCLUSIONnThese data indicate that u-PA and its interacting molecules, such as u-PAR and cathepsin B, may have some contributory effects on the metastatic potential of tumor cells in dedifferentiated parosteal osteosarcoma.

Antisense inhibition of cathepsin B in a human osteosarcoma cell line.

Tumor invasion and metastasis are complex, multistep processes involving cellular detachment from primary sites, adhesion to extracellular/cellular matrices, local proteolytic degradation of matrix proteins and migration. Several proteases are involved in these complex processes. Besides matrix metalloproteases (MMPs) (Liotta and StetlerStevenson, 1990) and the urokinase-type plasminogen activator (u-PA) (Schmidt et al 1992), cathepsin B (cat B) (Sloane et al. 1990) is considered to play important roles in the different destructive occurrences. Besides direct proteolysis of components of the basement membrane and extracellular matrix, cat B participates in enzyme activation cascades, including other cathepsins and uPA (Kobayashi et al 1991). To block not only the catalytic activity of the cysteine protease cat B (e.g. by anticatalytical antibodies), the osteosarcoma cell line MNNG/HOS was stably transfected with antisense cat B constructs, resulting in direct suppression of cat B expression in the tumor cells. We selected cell clones with the lowest enzyme expression to functionally characterize them in invitro assays of invasion and motility, compared with the parental cell line MNNG/HOS.