phosphorus-radioisotopes and Glioblastoma

Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action with promising brain tumor specificity. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP) - and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. The toxicity of ellipticine to U87MG glioblastoma cells and mechanisms of its action to these cells are aims of this study.. Ellipticine metabolites formed in U87MG cells were analyzed using HPLC. Covalent DNA modifications by ellipticine were detected by 32P-postlabeling. CYP enzyme expression was examined by QPCR and Western blot.. U87MG glioblastoma cell proliferation was efficiently inhibited by ellipticine. This effect might be associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP1A1, 1B1 and 3A4, lactoperoxidase and cyclooxygenase 1, the enzymes expressed in U87MG cells. Moreover, by inducing CYP1B1, 3A4 and 1A1 enzymes in U87MG cells, ellipticine increases its own enzymatic activation, thereby enhancing its own genotoxic and pharmacological potential in these cells. Ellipticine concentration used for U87MG cell treatment is extremely important for its pharmacological effects, as its metabolite profiles differed substantially predicting ellipticine to be either detoxified or activated.. The results found in this study are the first report showing cytotoxicity and DNA adduct formation by ellipticine in glioblastomas.

Topics: Antineoplastic Agents; Autoradiography; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; DNA Adducts; Dose-Response Relationship, Drug; Ellipticines; Glioblastoma; Humans; Phosphorus Radioisotopes; Polymerase Chain Reaction; RNA, Messenger

In vivo studies have demonstrated that pentavalent technetium-99m dimercaptosuccinic acid [(99m)Tc-(V)-DMSA] may be a useful tumour imaging agent. Several studies have suggested that (99m)Tc-(V)-DMSA uptake may be related to the structural similarity between the (99m)Tc-(V)-DMSA core and the PO(4)(3-) anion. As phosphate ions enter cells via NaPi cotransporters, we investigated whether (99m)Tc-(V)-DMSA uptake is mediated by NaPi cotransporters. (99m)Tc-(V)-DMSA and phosphate uptake kinetics were compared in three cancer cell lines (MCF-7, G152 and MG-63) under several conditions (with and without sodium and NaPi cotransporter inhibitor and at different pH). Determination of molecular NaPi cotransporter mRNA expression was performed by reverse-transcriptase polymerase chain reaction (Rt-PCR) assay. Results obtained in the presence of NaPi inhibitor, in sodium-free medium and at alkaline pH showed that (99m)Tc-(V)-DMSA accumulation is linked to NaPi cotransporter functionality. MCF-7 and G152 exhibited the same tracer uptake, whereas MG-63 showed the highest phosphate accumulation and the lowest (99m)Tc-(V)-DMSA uptake. These results were in accordance with mRNA NaPi expression, i.e. all cell lines expressed NaPi type III but MG-63 also co-expressed NaPi type I. The total level of NaPi cotransporter was highly correlated with phosphate accumulation, while the level of type III was related to (99m)Tc-(V)-DMSA uptake. We have demonstrated that (99m)Tc-(V)-DMSA uptake is specifically mediated by NaPi type III in cancer cells.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Glioblastoma; Humans; Hydrogen-Ion Concentration; Kinetics; Metabolic Clearance Rate; Neoplasms; Osteosarcoma; Phosphates; Phosphorus Radioisotopes; Potassium Compounds; Radionuclide Imaging; Radiopharmaceuticals; Sodium; Sodium-Phosphate Cotransporter Proteins; Sodium-Phosphate Cotransporter Proteins, Type I; Symporters; Technetium Tc 99m Dimercaptosuccinic Acid

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.

Topics: Chromosomes, Fungal; Chromosomes, Human; DNA Damage; DNA Repair; DNA, Neoplasm; Dose-Response Relationship, Radiation; Glioblastoma; Humans; Kinetics; Phosphorus Radioisotopes; Saccharomyces cerevisiae; Schizosaccharomyces; Tumor Cells, Cultured; X-Rays

Catalytic RNA or ribozymes have important potential applications as molecular biological tools in the study of gene expression and as therapeutic inhibitors of disease-causing genes. Very little is known, however, about the cellular uptake mechanisms of exogenously delivered synthetic ribozymes. In this study, we have characterized the uptake properties of a synthetic, 2'-O-methyl-modified ribozyme containing U4/U7 amino groups within the catalytic core of the hammerhead motif. The cellular uptake of the internally [32P]-radiolabeled hammerhead ribozyme in U87-MG glioma cells was temperature, energy, and pH dependent and involved an active process that could be competed with cold ribozyme of the same chemistry and sequence, an all 2'-O-methyl-modified ribozyme of the same sequence, antisense PS-ODNs, and a variety of other polyanions (salmon sperm DNA, spermidine, dextran sulfate, and heparin). Subcellular distribution studies of fluorescently labeled ribozymes confirmed an extranuclear, punctate localization similar to that observed for an endosomal marker, dextran. Our study highlights that hammerhead ribozymes, despite exhibiting a defined secondary structure, enter cells by an endocytic mechanism that appears to be similar to that reported for a variety of antisense ODNs. These observations should facilitate the development of more efficient delivery systems.

Topics: Base Sequence; Binding, Competitive; Biological Transport; Conserved Sequence; Dextran Sulfate; DNA; Endocytosis; Endosomes; ErbB Receptors; Glioblastoma; Heparin; Humans; Kinetics; Methylation; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides, Antisense; Phosphorus Radioisotopes; RNA, Catalytic; RNA, Messenger; Spermidine; Subcellular Fractions; Thionucleotides; Tumor Cells, Cultured

The immune complex kinase assay is the most widely applied method to assess the catalytic activity of protein tyrosine kinases. It offers the advantage that the activity of a single selected enzyme can be determined, and that the enzyme activity can be normalized for the amount of enzyme in a parallel immunoblotting experiment. Here, we describe the use of the recently introduced isotope phosphorus-33 for the protein kinase assay. The lower energy of 33P, compared with the traditionally applied 32P, allows the simultaneous examination of the amount of enzyme with 125I-labeled antibodies. By analysing one and the same sample for both kinase activity and protein amount, the variation between parallel processed samples is avoided. Using this method, specific kinase activities can be calculated with high precision. The assay is particularly useful for the detection of cytokine and growth factor-induced activation of kinases, as changes in enzyme amounts by subcellular relocalization can be distinguished.

Topics: Carcinoma; Glioblastoma; Humans; Immunoblotting; Methods; Neoplasm Proteins; Phosphorus Radioisotopes; Phosphotyrosine; Precipitin Tests; Protein-Tyrosine Kinases; Sensitivity and Specificity; Skin Neoplasms; Tumor Cells, Cultured

140 patients with brain tumor were treated by CT-guided stereotactic injections of radionuclides, such as Aurum-198 (198 Au), Phosphorus-32 (32 P) and Yttrium-90 (90Y). Of these patients aged from 3 to 67 years (average 37), 64 were male and 76 female. Astrocytoma was found in 75 patients, craniopharyngioma in 46, metastatic carcinoma in 7, meningioma in 5, germinoma in 4 and pituitary adenoma in 3. The tumors were located in the deep part or functionally critical area of the brain. After 267 times of injection of colloidal isotopes, no major adverse effects or complications occurred. Follow-up for 6 to 48 months showed improvement in symptoms in 104 (74.3%) patients and CT scanning showed the diminished tumors.

Topics: Adolescent; Adult; Aged; Astrocytoma; Brachytherapy; Brain Neoplasms; Child; Child, Preschool; Craniopharyngioma; Female; Glioblastoma; Gold Radioisotopes; Humans; Male; Middle Aged; Phosphorus Radioisotopes; Stereotaxic Techniques; Tomography, X-Ray Computed; Yttrium Radioisotopes

Instillation of [32P]chromic phosphate to cystic brain tumors was performed in six patients. Three patients had craniopharyngioma, two had Grade IV astrocytoma and one had Grade II astrocytoma. The cyst volumes ranged from 2 to 44 cc. A calculated dose of 20,000 rad was delivered to the cyst wall. The [32P]chromic phosphate dose given to achieve this dose ranged from 0.11 mCi to 2.5 mCi. Radionuclide leakage was not detected in either the central nervous system or the reticuloendothelial system by bremsstrahlung scanning. Stereotactic instillation was done in some cases, others had indwelling catheters. The frequency of cyst fluid aspiration in the three patients with craniopharyngioma decreased postinstillation. In the two patients with Grade IV astrocytoma, reductions in both the CT documented cyst size as well as the frequency of cyst aspiration were noted. We conclude that [32P]chromic phosphate installation by stereotactic or indwelling catheter method is a safe and helpful procedure in the management of cystic brain tumors.

Topics: Adult; Aged; Brain Neoplasms; Craniopharyngioma; Female; Glioblastoma; Humans; Male; Middle Aged; Phosphorus Radioisotopes; Pituitary Neoplasms