Radiosensitization by histone deacetylase inhibition in an osteosarcoma mouse model.

BACKGROUND: Osteosarcomas (OS) are highly malignant and radioresistant tumors. Histone deacetylase inhibitors (HDACi) constitute a novel class of anticancer agents. We sought to investigate the effect of combined treatment with suberoylanilide hydroxamic acid (SAHA) and radiotherapy in OS in vivo. METHODS: Clonogenic survival of human OS cell lines as well as tumor growth delay of OS xenografts were tested after treatment with either vehicle, radiotherapy (XRT), SAHA, or XRT and SAHA. Tumor proliferation, necrosis, microvascular density, apoptosis, and p53/p21 were monitored by immunohistochemistry. The CD95 pathway was performed by flow cytometry, caspase (3/7/8) activity measurements, and functional inhibition of CD95 death signaling. RESULTS: Combined treatment with SAHA and XRT markedly reduced the surviving fraction of OS cells as compared to XRT alone. Likewise, dual therapy significantly inhibited OS tumor growth in vivo as compared to XRT alone, reflected by reduced tumor proliferation, impaired angiogenesis, and increased apoptosis. Addition of HDACi to XRT led to elevated p53, p21, CD95, and CD95L expression. Inhibition of CD95 signaling reduced HDACi- and XRT-induced apoptosis. CONCLUSION: Our data show that HDACi increases the radiosensitivity of osteosarcoma cells at least in part via ligand-induced apoptosis. HDACi thus emerge as potentially useful treatment components of OS.

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PURPOSE: Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. METHODS: Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. RESULTS: SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. CONCLUSION: SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

High cure rates and reduced long-term toxicity in pediatric Hodgkin's disease: the German-Austrian multicenter trial DAL-HD-90. The German-Austrian Pediatric Hodgkin's Disease Study Group.

PURPOSE: To further reduce therapy-related late effects in patients with pediatric Hodgkin's disease (HD) while maintaining the high cure rates achieved with vincristine, prednisone, procarbazine, and doxorubicin (OPPA) or OPPA/cyclophosphamide, vincristine, prednisone, and procarbazine (COPP) chemotherapy and involved-field radiotherapy. The risk of testicular dysfunction was addressed by substituting etoposide for procarbazine (OEPA) in the induction therapy for boys. Radiation doses and fields were further reduced. PATIENTS AND METHODS: Three hundred nineteen boys and 259 girls younger than 18 years with previously untreated HD, enrolled onto the study between 1990 and 1995, were allocated to treatment group (TG)1 (early stages), TG2 (intermediate stages), or TG3 (advanced stages). All groups underwent two cycles of OEPA (boys) or OPPA (girls) for induction chemotherapy. TG2 and TG3 continued on additional two or four cycles, respectively, of COPP. Low-dose radiotherapy was given to the initially involved sites, ie, reduced involved fields. RESULTS: Initial response to OPPA or OEPA induction was virtually identical. Eight of 578 patients experienced early progression of HD. Thirty-seven relapses, three secondary tumors, and no secondary leukemias have been recorded, with a median follow-up duration of 5.1 years (maximum, 8.1 years). Thirteen of 578 patients died. The probability of 5-year event-free survival/overall survival is 91%/98% in the total group, 94%/97% with OPPA, and 89%/98% with OEPA induction therapy. Risk factor analysis showed two significant prognostic factors: histologic subtype NS2 and "B" symptoms. OEPA induction therapy, large mediastinal tumor, and age were not significant. Preliminary studies of testicular function indicate a lower risk of germ cell damage than previously documented with OPPA. CONCLUSION: OEPA is a satisfactory alternative to OPPA. Radiotherapy can be confined to involved sites when combined with appropriate chemotherapy. The DAL-HD-90 regimen represents a comprehensive treatment program for all stages of pediatric HD and offers a favorable benefit/risk ratio, combining excellent disease control, moderate acute toxicity, and reduced long-term toxicity.

Complete sequence of glycolytic enzymes in the mycorrhizal basidiomycete, Suillus bovinus.

Axenic cultures of Suillus bovinus were cultivated in inorganic liquid medium with glucose as a carbon source at 25 degrees C and continuous supply of oxygen by aeration with compressed air in the dark. Exogenous fructose as sole carbon source yielded about 50% less increase in dry weight than glucose. This resulted from different uptake velocities. Sucrose as sole exogenous carbon source yielded no measurable increase in dry weight. In glucose cultures, activities of all glycolytic enzymes were found. Maximum specific activities varied largely (from about 60 [fructose 6-phosphate kinase] to about 20,000 [triosephosphate isomerase] nmoles.mg protein-1.min-1). Apparent K(m)-values also varied over more than two orders of magnitude (0.035 mM [pyruvate kinase] to 6.16 mM [triosephosphate isomerase]). Fructose 6-phosphate kinase proved to be the fructose 2,6-bisphosphate-regulated type, aldolase the divalent cation-dependent (class II) type and glyceratephosphate mutase the glycerate 2,3-phosphate-independent type of the respective enzymes. Eight of the 10 enzymes exhibited pH-optima between 7.5-8.0. Triosephosphate isomerase and pyruvate kinase showed highest activities at pH 6.5. Regulatory sites within the glycolytic pathway of Suillus bovinus are discussed; fructose 6-phosphate kinase appears to be its main bottle neck.

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.

Obesity and hypertestosteronaemia are independently and synergistically associated with elevated insulin concentrations and dyslipidaemia in pre-menopausal women.

The association of obesity and hypertestosteronaemia with elevated insulin concentration and dyslipidaemia was studied in 15 non-obese and 15 obese, hypertestosteronaemia patients; 14 non-obese and 10 obese, normotestosteronaemic subjects served as controls. Data were subjected to multivariate analysis. Enhanced body mass index (BMI kg/m2) resulted in a significant elevation of basal insulin (b-Ins), glucose-stimulated (delta) insulin (del-Ins), triglycerides (TG), very low density lipoprotein (VLDL), low density lipoprotein (LDL), and LDL/high density lipoprotein (HDL) ratio, and in a significant reduction of HDL. Furthermore, it was shown that BMI was positively correlated with TG, VLDL, LDL and LDH/HLD ratio, and negatively correlated with HDL in the normotestosteronaemic groups. Hypertestosteronaemia was associated with a significant increase of del-Ins, VLDL and LDL/HDL ratio, and with a significant decrease of HDL concentration. Testosterone was directly associated with del-Ins and LDL/HDL ratio, and inversely related to HDL in the non-obese groups. Summation effects of obesity and hypertestosteronaemia were found for del-Ins and VLDL. The data suggest that obesity and hypertestosteronaemia are independently and jointly associated with insulin resistance and dyslipidaemia, indicating an increased risk for coronary heart disease. The highest risk rate was found in obese hypertestosteronaemic patients. Serum testosterone may be a useful marker in detecting metabolic disorders connected with cardiovascular risk.