Inhibitory effects of antisense cathepsin B cDNA transfection on invasion and motility in a human osteosarcoma cell line.

Increased activity, membrane association, and secretion of cathepsin B have been shown to correlate positively with invasiveness and the metastatic properties of many tumor entities. Cathepsin B is able to directly facilitate invasion by degrading extracellular matrix components or to indirectly facilitate invasion by activating other matrix-degrading proteases like the urokinase-type plasminogen activator. To investigate the role of cathepsin B in bone tumor invasion, the osteosarcoma cell line MNNG/HOS was stably transfected with an expression vector capable of expressing the antisense cDNA transcript of cathepsin B. Five stably transfected antisense cell clones, the control (vector) cell clones, and the parental cells were characterized. At first, the stable incorporation of the constructs was demonstrated by Southern blot analysis. In ELISA assays, all antisense clones showed a significant reduction at the cathepsin B antigen level (about 70%) as compared with the control cell clones and MNNG/HOS. Similar results were obtained for cathepsin B activity in the antisense-transfected cells. In the antisense cell clones, Northern blot analysis and reverse transcription-PCR revealed a considerable decrease of approximately 50% in the levels of cathepsin B mRNA. Expression of cathepsins L and K (sequence homologies) was not affected. The invasive potential and migration of untransfected and transfected tumor cell clones in vitro were analyzed in Transwell chambers. Antisense-transfected cells showed a markedly lower invasion and motility than did MNNG/HOS and the controls. Adhesion to collagen I and matrigel matrices was not affected. These results demonstrate that cathepsin B is involved in the complex proteolytic processes in invasive osteosarcomas.

Prognostic relevance of p53 alterations and Mib-1 proliferation index in subgroups of primary liposarcomas.

For prognostic analyses of p53 alterations (p53 gene mutations + p53 immunopositivity) and Mib-1 proliferation index, we investigated 42 primary malignant lipomatous tumors for which complete clinical data and a long follow-up were available. p53 gene mutations were investigated by PCR-single strand conformation polymorphism-sequencing analysis, and immunohistochemistry was used to determine p53 protein expression and Mib-1 proliferation index. We found a mutation frequency of 14.3%. Nine liposarcomas (21%) were p53 immunopositive, and 11 (26.2%) had at least one p53 alteration. In myxoid liposarcomas, p53 alterations are not relevant to the presence or absence of round cell components. Pleomorphic liposarcomas showed a significantly higher proliferation index and more p53 alterations than myxoid or well-differentiated variants (P<0.001). When the Cox's regression analysis tumors of grade III histology (P = 0.005) was performed, the pleomorphic subtype (P = 0.016) and liposarcomas of retroperitoneal localization (P = 0.015) showed a significantly poorer prognosis. Moreover, we found that p53 alterations and high proliferation index correlated significantly with reduced overall survival. Their prognostic value seemed to be higher in myxoid than in pleomorphic liposarcomas. The metastasis-free survival was reduced in patients who had liposarcomas with p53 alterations (P = 0.171) or elevated proliferation index (P<0.016), reflecting a more aggressive behavior. In conclusion, the determination of p53 alterations and/or Mib-1 proliferation index is useful for assessing the prognosis of patients with liposarcomas and may especially be helpful in dividing different prognostic groups for patients with myxoid variants.

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-1beta), IL-6 and tumor necrosis factor alpha (TNF-alpha) as well as the anti-inflammatory cytokine transforming growth factor beta (TGF-beta) 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-1beta significantly stimulated APN mRNA expression in both cell lines, it influenced the enzyme activity only in MG63. TNF-alpha and TGF-beta, 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.

Gene alterations at the CDKN2A (p16/MTS1) locus in soft tissue tumors.

The CDKN2A gene (p16/MTS1) is a tumor suppressor that is frequently deleted, mutated, or inactivated by transcriptional silencing in certain tumor types and many tumor cell lines. We analyzed CDKN2A gene mutations and the frequency of loss of heterozygosity (LOH) at the CDKN2A locus in 135 soft tissue sarcomas. PCR-SSCP analysis of exons 1 and 2 of CDKN2A gene revealed only one missense mutation in codon 15 in a rhabdomyosarcoma. LOH-analysis was performed with two polymorphic markers in the surrounding regions of the CDKN2A gene (D9S171, D9S162) and the sequence-tagged-site marker c5.1. An allelic loss was found in 7/135 cases (5.1%) and was a characteristic of poorly differentiated sarcomas. We observed a high frequency of microsatellite instability expressed as allelic imbalances (25%). Presumably, alterations of the CDKN2A gene do not contribute to the oncogenesis in the majority of soft tissue tumors.

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.

Expression of cathepsin K in chordoma.

Invasive growth of chordoma is accompanied by severe destruction of adjacent bone tissue, a fact that requires high proteolytic activity at the tumor invasion fronts. In this context, cathepsin K is a candidate molecule. It is a protease with high collagenolytic and elastinolytic activity and previously thought to be restricted to osteoclasts and osteoclast-mediated bone resorption. In this study, 44 cases of chordoma of sphenooccipital localization, and 10 embryo-fetal specimens including chorda dorsalis were studied immunohistochemically for their expression of cathepsin K. In 4 additional snap-frozen chordoma cases, the enzyme expression was investigated by reverse transcription polymerase chain reaction and enzyme histochemistry. Ten chondrosarcomas of the skull base served as controls. Various concentrations of cathepsin K mRNA could be seen in all snap-frozen chordoma specimens. The protease was immunohistochemically expressed by the tumor cells. The immunoreactions were accentuated at the tumor invasion fronts. Enzyme histochemistry indicated a strong tumor cell-associated cathepsin K activity in invasive tumor components. In contrast to chordoma, cathepsin K was not significantly expressed in chorda dorsalis and chondrosarcoma of the skull base. In chondrosarcoma, protease expression was limited to osteoclastic cells localized between infiltrative tumor components and regular bone trabeculae. This study shows the significant expression and activity of cathepsin K in chordoma and implicates an important and direct role of this protease in the infiltrative growth of this tumor. This protease expression occurred during neoplastic transformation and did not appear in chorda dorsalis.

Inducible nitric oxide synthase expression in human urinary bladder cancer.

Nitric oxide (NO) is generated by a family of enzymes, nitric oxide synthases (NOS), in a wide range of mammalian cells. NO produced by the inducible NOS isoform (iNOS) has been suggested to play an important role in tumor biology with both tumor promoter and anti-tumor activity. Here, the cellular localization of iNOS in tissue of 100 cases of urinary bladder cancer was assessed immunohistologically using a commercially available antiserum. Positive iNOS immunostaining was detected in all samples of tumor tissue, whereas nonmalignant tissue adjacent to malignant areas did not show any iNOS positivity. The tumor tissue revealed a highly inhomogeneous staining pattern. In addition to uniformly stained tumor specimens, we also found markedly iNOS-positive tumor islets in the midst of unstained tumor tissue and scattered individual tumor cells expressing marked staining. In some cases, the tumor tissue showed no or only weak staining intensity. In some instances, the superficial epithelial layer of papillary carcinomas was extremely immunoreactive, in other cases it was not. Thus we were unable to show a clear correlation to tumor grade or stage. Further studies with a diversity of tumor markers including molecular genetics techniques will be necessary to elucidate how and to what extent NO and bladder cancer of different grades and stages are functionally interrelated.

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

Alterations 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.

Protease expression in dedifferentiated parosteal osteosarcoma.

BACKGROUND: Parosteal 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. METHODS: The 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. RESULTS: Both 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. CONCLUSION: These 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 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.

[Antisense inhibition of urokinase in a human osteosarcoma cell line]. / Antisense Inhibition von Urokinase in einer humanen Osteosarkomzellinie.

In the multistep process of tumor metastasis, different cellular functions are known to be influenced by the urokinase plasminogen activator (u-PA). In different types of malignancies, u-PA has been shown to correlate strongly with a malignant tumor cell phenotype. Besides its proteolytic activity, the enzyme is effective by signal transduction mechanisms. To elucidate u-PA functions in osteosarcoma, in the present study, the osteosarcoma cell line MNNG/HOS was transfected with an antisense (as) expression vector encoding the 3' end of u-PA-cDNA. Several stably transfected cell clones were characterized and compared with the parental cell line. The antisense transfection resulted in: (1) stable incorporation of the vector construct into cellular genome, as demonstrated in Southern blot; (2) decreased u-PA expression in Northern blot; (3) 50% reduced u-PA protein expression in both cell homogenate and cell culture medium; (4) unchanged cellular proliferation and u-PAR (urokinase plasminogen activator receptor) expression. In comparing functional analysis, as-clones showed (I) significant reduced in vitro invasion and motility (chemotaxis with collagen I); (II) significantly reduced adhesion activity to both vitronectin and collagen I matrices but unchanged adhesion on matrigel; (III) reduced in vivo metastasis in chick embryos after i.v.-application. All together, this data show that malignancy of the osteosarcoma cell line MNNG/HOS is positively influenced by urokinase in terms of migration and selective adhesion. Both effects were observed besides the previously described enzyme functions in tumor cell invasion and basement membrane degradation.

Malignant peripheral nerve sheath tumor of the esophagus.

BACKGROUND: Sarcomas of the esophagus are rare representing 0.1-1.5% of all esophageal tumors. We report a case of malignant peripheral nerve sheath tumor (MPNST) of the esophagus in a 60-year-old woman. METHODS: The diagnosis was made preoperatively on endoscopic biopsy and confirmed after tumor resection by immunohistochemistry as well as electron microscopy. The patient underwent abdominal-thoracicen bloc esophagectomy with mediastinal lymphadenectomy and intrathoracic esophagogastrostomy. RESULTS: Our therapeutic concept for the first case of a high-grade MPNST (malignant schwannoma) of the esophagus resulted in a recurrence-free interval of 4 years. CONCLUSION: The therapy of choice was abdominal-thoracic en bloc esophagectomy with tumor-free resection margins and esophageal reconstruction with the stomach. After R0-resection we found no indication for adjuvant radio- and/or chemotherapy.

Improved detection of p53 mutations in soft tissue tumors using new gel composition for automated nonradioactive analysis of single-strand conformation polymorphism.

We report a new nonradioactive method to detect sequence changes, including single-base substitutions through shifts in electrophoretic mobility using an automated fluorescence sequencer (ALFexpress, Pharmacia, Biotech) connected to external cooling equipment. Single strands were identified by incorporation of fluorescein-labeled primers during amplification and subsequent laser detection at the bottom of the gel. The amplified polymerase chain reaction (PCR) products were heat-denatured and loaded onto a polyacrylamide gel under nondenaturing conditions and strict control of constant low temperature. Peak shifts in the fluorogram indicated mutations. A novel gel composition improved the detection rate for mutations considerably. Automatic analysis of single-strand conformation polymorphism (SSCP) gels saves time and costs, and is highly reproducible. The method was applied for mutation screening in exon 7 of the p53 tumor suppressor gene in DNA of freshly frozen soft tissue tumors. The mutation spectrum and frequency in exon 7 of the p53 gene are discussed with respect to oncogenesis in soft tissue sarcomas.

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.

[Telomere lengths and telomerase activity in liposarcomas]. / Telomerlängen und Telomeraseaktivität in Liposarkomen.

We measured telomerase activity in 36 malignant and seven benign lipomatous neoplasias from 34 patients to assess the role of telomerase in the development of liposarcoma. The sensitive PCR-based telomerase assay (telomeric repeat amplification protocol-TRAP) was applied. We correlated telomerase activity with the shortening or elongation of telomeric repeat fragment length (TRF), measured by using hybridization with a telomere specific oligonucleotide probe. Telomerase activity was demonstrated in 69% of malignant tumors. This information may be helpful in distinguishing benign tumors from malignant neoplasias. Telomerase expression, however, seems to be characteristic of poorly differentiated liposarcomas. Telomerase activity was not correlated with age at the time of diagnosis or with sex. We observed that telomerase expressing tumors had higher proliferation indices than neoplasias lacking telomerase. Telomerase activity was observed in all eight recurrences, suggesting a close association of telomerase with the biologic behavior of liposarcomas. Therefore, we assume that telomerase plays a key role in the establishment and progression of lipomatous tumors.

Induction of TFF1 gene expression in pancreas overexpressing transforming growth factor alpha.

BACKGROUND/AIMS: Chronic pancreatitis is an inflammatory disease of the exocrine pancreas associated with extensive fibrosis, enlarged pancreatic ducts, acinar cell degeneration, and the formation of tubular complexes. The molecular and biochemical alterations associated with these histological changes are not kown. Generally, the new family of TFF peptides (formerly known as P-domain peptides or trefoil factors) is aberrantly expressed during chronic inflammatory diseases of the gastrointestinal tract. METHODS: Using human pancreatic tissues obtained from patients with chronic pancreatitis and murine pancreatic tissues obtained from transgenic mice overexpressing transforming growth factor alpha (TGF-alpha), the expression and cellular distribution of TFF1 was analysed using northern blot analysis, polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: In the normal human pancreas, TFF1 was scarce, with only a few ducts exhibiting cytoplasmic TFF1 immunoreactivity. In contrast, human chronic pancreatitis tissue specimens exhibited strong TFF1 immunoreactivity in ductal cells, areas of ductal hyperplasia, and tubular complexes. Semiquantitative PCR analysis of TFF1 mRNA levels showed enhanced expression of TFF1 in the pancreas of patients with chronic pancreatitis. Furthermore, TFF1 mRNA levels were detectable in the pancreas in four of five transgenic mice overexpressing TGF-alpha. In contrast, four of five wild type mice did not exhibit a TFF1 mRNA transcript. In addition, while no specific TFF1 immunoreactivity was present in the pancreas of the wild type mice, ductal epithelial cells and duct-like tubular complexes in the pancreas of the transgenic mice overexpressing TGF-alpha exhibited pronounced TFF1 immunoreactivity. CONCLUSIONS: Ductal cells and tubular complexes in pancreatic fibrosis express TFF1. As the 5'-flanking region of TFF1 contains an epidermal growth factor responsive enhancer region and the expression of epidermal growth factor and TGF-alpha is enhanced in pancreatic fibrosis, the enhanced expression of TFF1 in pancreatic fibrosis may be mediated by TGF-alpha.