Effects of hyperthermia on heat shock protein expression, alkaline phosphatase activity and proliferation in human osteosarcoma cells.

Hyperthermia can be used as a possible adjuvant therapy in treatment of cancer patients. In this study, the direct effect of hyperthermia on osteosarcoma derived cell lines HOS85, MG-63 and SaOS-2 was investigated. Heat shock at 42 degrees C inhibited proliferation significantly in all three cell lines tested. Furthermore a sub-lethal heat shock (42 degrees C, 1 h) decreases alkaline phosphatase activity, the absolute marker for osteoblast-like cells, in all of the three cell lines. Hsp70 was expressed constitutively and was found to be upregulated in a time-dependent manner; by up to 150% in Western blot analysis. The results of this study indicate that heat shock has an inhibitory effect on human osteosarcoma cells. These data suggest that hyperthermia has an anti-tumour effect on cancers of the bone and might, therefore, become an adjuvant treatment option.

In vivo and in vitro cellular ingrowth into a new generation of artificial ligaments.

Artificial ligaments are a useful tool in ligament reconstruction. Although the new generation of artificial ligaments shows encouraging clinical results, in contrast to earlier generations studies on the biological properties are lacking. Biopsies were taken from a ligament advancement reinforcement system (LARS) 6 months after implantation and investigated by histochemistry. An in vitro study seeding human fibroblasts or osteoblast-like cells (up to 10(6) cells for 21 days) on ligament pieces (5 x 5 mm) was conducted and analyzed by histochemistry. The biopsies showed complete cellular and connective tissue ingrowth in the LARS ligament. In vitro fibroblasts and osteoblast-like cells encapsulated the fibers by building a cellular net around them. To our knowledge, these findings demonstrate for the first time the cellular ingrowth into the LARS ligament. This mechanism might explain the strength and the inert behavior of the ligament without the synovialitis shown in clinical studies.

Serum levels of TNF-beta and sTNF-R in patients with malignant bone tumours.

OBJECTIVE: To determine serum levels of TNF-beta and soluble TNF-R in patients with primary highly malignant bone tumours. PATIENTS AND METHODS: Sera of 27 patients with highly malignant osteosarcoma and Ewing sarcoma were taken at the time of diagnosis and analysed by ELISA. RESULTS: Both TNF-beta and sTNF-R levels were lower in sera from osteosarcoma patients as compared to those from Ewing sarcoma. In patients with high-grade osteosarcoma, but not Ewing sarcoma, high levels of TNF-beta correlated with bad response to neoadjuvant chemotherapy. CONCLUSION: In patients with high-grade osteosarcoma TNF-beta levels seem to be of predictive value and both TNF-beta and sTNF-R seem to be of diagnostic value for differentiation between high-grade osteosarcoma and Ewing sarcoma.

Suramin suppresses growth, alkaline-phosphatase and telomerase activity of human osteosarcoma cells in vitro.

Neoadjuvant chemotherapy in osteosarcoma improves the survival dramatically, but there is currents drug resistance in about 25% of patients, leading researchers to investigate alternative therapy forms. Suramin has in the last two decades been used as salvage therapy in some cancers. This study was undertaken to investigate suramin as a possible salvage therapy in osteosarcoma. The effect of suramin on three human osteosarcoma cell lines (MG-63, HOS and SaOS-2) and three primary osteosarcoma cell lines isolated from biopsies was investigated. Suramin significantly inhibited cell proliferation, determined by 3H-thymidine incorporation, of osteosarcoma cells at a dose ranging from 250 to 500 microg/ml. Suramin decreased the secretion of alkaline-phosphatase after stimulation by 1,25-dihydroxy-Vitamin D(3) up to 50% and decreased telomerase activity by up to 40%. The data demonstrate that suramin has marked in vitro effects on human osteosarcoma cells supporting further clinical investigation.

Serum concentrations of APO-1/Fas and interleukin-1beta-converting enzyme in osteosarcoma correlate with response to chemotherapy.

BACKGROUND: Cytotoxic drugs used in neoadjuvant chemotherapy may exert their effect by activation of apoptosis. Both APO-1/Fas and Interleukin-1beta-converting (ICE)/caspase-1 are believed to act as mediators of apoptotic cell death. PATIENTS AND METHODS: The sera of 21 patients with high-grade osteosarcoma were tested at the time of diagnosis by ELISA and compared with clinical data. RESULTS: The mean APO-1 serum levels were 844 +/- 344 pg ml(-1) and the mean ICE serum levels were 212 pg ml(-1) +/- 155 pg ml(-1) for all patients. Furthermore, both serum levels were found to be statistically significantly higher in patients with good regression grades compared to those in patients with bad regression grades. CONCLUSION: In highly-malignant osteosarcoma elevated serum levels of both APO-1 and ICE seem to be predictive of good response to chemotherapy.

Hyperthermia increases the susceptibility of chondro- and osteosarcoma cells to natural killer cell-mediated lysis.

As an adjuvant to chemotherapy hyperthermia has proven to be successful as a treatment for osteo- and chondrosarcoma patients. The aim of this study was to investigate whether hyperthermia could increase cellular expression of heat-shock-protein 72 in human osteo- and chondrosarcoma cells and how heat treatment would affect their susceptibility to natural killer cell (NK-cell)-mediated lysis. About 5-10% of the peripheral mononuclear blood cells (PBMC) in the human peripheral blood are natural killer cells (NK-cells). Natural cytotoxicity, mediated by NK-cells, is believed to play an important role in host defense against cancer. The exact mechanisms of recognition of target cells and subsequent NK-cell activation are not yet known. NK-cells, isolated from the peripheral blood of healthy donors, were enriched by magnetic cell-separation to a purity of 85-97%, assessed by FACS-analysis. The susceptibility of heat-treated (42.5 degrees C, 90 minutes) and untreated osteosarcoma (MG63) and chondrosarcoma (HTB94) cell lines to NK-killing was determined by a release assay of lactate dehydrogenase (LDH). Lysis by NK-cells was increased by heat treatment of the target cells from 16.6% + 4.5% to 33% + 15%, p=0.035, for osteosarcoma cells, (E/T ratio of 5:1) and from 13.7% + 3.1% to 27.9% + 16.9%, p=0.021, (E/T ratio of 20:1) for chondrosarcoma cells. An increased expression of HSP72 of chondro- and osteosarcoma cells after heat treatment was detected by the Western blot technique. The results of this study show that hyperthermia increases HSP72 expression in osteo- and chondrosarcoma cells and their susceptibility to NK-cell-mediated lysis. These findings may lead to new therapeutic strategies, using hyperthermia to improve immunological defense against chondro- and osteosarcoma cells.

Heat shock protein expression in the transplanted human kidney.

Heat shock proteins (HSPs) have been shown to represent potential target molecules for T-cell-mediated allograft rejection in heart and kidney transplants. In the present study, we therefore investigated the expression of HSP subtypes 60, 72, and 73 in normal kidneys and qualitative and/or quantitative changes in rejected renal allografts. Six normal kidney tissue specimens, three biopsies from patients with minimal change nephritis, as well as 37 biopsies and eight transplant nephrectomy specimens of patients with renal allograft rejection were studied. Type and severity of rejection were assessed according to the Banff classification. Immunohistochemical demonstration of HSP expression was performed using specific monoclonal antibodies after wet autoclave antigen retrieval on sections from either Carnoy-fixed (biopsies) or formalin-fixed (transplant nephrectomies) and paraffin-embedded tissue. The expression was scored in a semiquantitative manner. All three subtypes were found to be constitutively expressed in normal kidney tissue and in noninflammatory minimal change nephritis, albeit with a characteristic compartmental and cellular distribution. Rejection resulted in a higher immunohistochemical scoring for all three HSP subtypes in compartments in which they were normally present; in addition, a de novo expression of HSP60 was found in the vascular compartment and, moreover, infiltrating mononuclear cells were strongly immunoreactive for HSP60 and HSP73. Only quantitative differences were observed for HSP72 immunoreactivity. These results indicate that rejection episodes are paralleled by an increased but differential expression of HSPs in the glomerular, tubular, and vascular compartments of the kidney. This enhancement as well as the de novo appearance of HSP60 on vascular endothelial cells might explain the presence of HSP-reactive T lymphocytes in rejected allografts.

Heat shock protein 72 expression in chondrosarcoma correlates with differentiation.

PURPOSE: Heat shock proteins (hsp) are involved in tumor immunity, and a correlation with survival, occurrence of metastases, and drug resistance has been reported. It was the aim of this study to investigate the expression of heat shock proteins in chondrosarcomas and chondromas. METHODS: Hsp expression was investigated immunohistochemically on paraffin-embedded sections of 37 consecutive patients (24 male and 13 female, mean age 48 years) with chondrosarcoma and of ten patients (six male, four female, mean age 36 years) with chondroma. RESULTS: Chondromas showed a positive staining for hsp27 in 100%, for hsp60 in 30%, for hsp72 in 80%, for hsp73 in 80%, and for hsp90 in 90%. In chondrosarcoma a decreased expression was found for hsp27 (62% positive, P<0.05) and hsp72 (43% positive, P<0.05), whereas no significant difference to chondromas was detected in the expression of hsp60 (49% positive), hsp73 and hsp90 (73% and 81% positive, respectively). In addition, hsp72 expression showed a correlation with differentiation of the tumors (P<0.05); the lowest hsp72 expression was found in G3 chondrosarcomas (only 13% positive). No correlation with respect to differentiation was found for the expression of the other hsps. CONCLUSIONS: This study shows a different expression of hsps in chondrosarcomas and chondromas. Together with the correlation of hsp72 expression with low differentiation, this finding could lead to new experimental and diagnostic strategies.