MG Human Glioma Research Articles

Gangliosides have a bimodal effect on DNA synthesis in U-1242 MG human glioma cells.

GM1, GD1a, and GT1b inhibit both PDGF-stimulated and serum-stimulated DNA synthesis in Swiss 3T3 cells and the human glioma cell line U-1242 MG in a dose-dependent manner. The ganglioside inhibitory effect is counteracted in a dose-responsive fashion by serum such that ganglioside-induced inhibition is essentially abolished in 10% serum. Because of the potentially important role that gangliosides play in growth regulation of human gliomas, this phenomenon was studied in detail using U-1242 MG cells. Stimulation of DNA synthesis by low doses of serum in U-1242 MG cells is inhibited in a dose-responsive fashion by ganglioside GM1. However, serum itself counteracts the inhibitory effect of ganglioside in a dose responsive way. Kinetic analyses demonstrate that GM1 competes with some components of serum for sites on U-1242 MG cells (Kb of GM1 = 12.5 microM). On the other hand, GM1, GD1a, and GT1b stimulate DNA synthesis in quiescent U-1242 MG cells in both sparse and confluent conditions, indicating that ganglioside-stimulated DNA synthesis is dependent on the phase of cellular growth rather than cellular density. This growth stimulatory effect of gangliosides is more potent on quiescent, confluent cells than quiescent, sparse cells. These results demonstrate that exogenously added gangliosides can have opposite (bimodal) effects on progression of human glioma cells through the cell cycle depending upon the growth phase of the cells.

Rapid intracellular assembly of tenascin hexabrachions suggests a novel cotranslational process.

Tenascin, an extracellular matrix protein that modulates cell adhesion, exists as a unique six-armed structure called a hexabrachion. The human hexabrachion is composed of six identical 320 kDa subunits and the structure is stabilized by inter-subunit disulfide bonds between amino-terminal segments. We have examined the biosynthesis of tenascin and its assembly into hexabrachions using pulsechase labeling of U-138 MG human glioma cells. Newly synthesized tenascin hexamers are secreted within 60 minutes of translation initiation. Intracellularly, as early as full length tenascin can be detected in pulse-labeled cell lysates, it is already in hexameric form. No precursors, such as monomers, dimers, or trimers, were identified that could be chased into hexamers. This lack of assembly intermediates suggests that nascent tenascin polypeptides associate prior to completion of translation. In contrast, fibronectin monomers in the same lysates are gradually formed into disulfide-bonded dimers. Although hexamer assembly is rapid, the rate-limiting step in secretion appears to be transport to the medial Golgi as endoglycosidase H-resistance was not detected until after a 30 minute chase. These results provide evidence for a novel co-translational mechanism of tenascin assembly which would be facilitated by its length and by the amino-terminal location of the assembly domain.

Detection of neutralizing antibodies against human T-cell leukemia virus type 1 using a cell-free infection system and polymerase chain reaction.

We have developed a cell-free infection system to titrate neutralizing antibodies against human T-cell leukemia virus type 1 (HTLV-1) using the polymerase chain reaction (PCR). S+L-CCC (8C) feline kidney or U-251 MG human glioma cells were infected with a cell-free culture supernatant derived from HTLV-1-infected c77 feline cells. DNA was extracted from 8C or U-251 MG cells after incubation for 24 hr and amplified by PCR. The c77 cell supernatant gave discrete bands, whereas those of HTLV-1-positive T cells did not. When the inocula were treated with HTLV-1 antibody-positive human sera or the monoclonal or polyclonal antibody against the peptide 190-199 of HTLV-1 envelope protein gp46, the subsequent formation of HTLV-1 proviral DNA was inhibited. We determined the titers of neutralizing antibodies by densitometrically scanning the intensity of the PCR bands. These titers correlated well with those determined by the plaque assay using a pseudotype of vesicular stomatitis virus bearing the envelope antigens of HTLV-1. At high serum concentrations, many seronegative samples markedly inhibited the plating of the HTLV-1 pseudotype whereas they barely affected results obtained by PCR. Thus, the c77-PCR system can detect neutralizing antibodies against HTLV-1 even at low titers.

Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27

We have found that the small stress protein, hsp27, exists in extracts of U251 MG human glioma cells in two forms: a large or aggregated form (L-hsp27, 300-400 kDa) and a small or dissociated form (S-hsp27, < 70 kDa), as indicated by centrifugation on sucrose density gradients. Dissociation of L-hsp27 to S-hsp27 was enhanced by incubation of cells with phorbol 12-myristate-13 acetate, interleukin-1 alpha, tumor necrosis factor alpha, or okadaic acid, all of which are known to enhance or mimic the effects of phosphorylation of hsp27 without stimulation of its synthesis. Exposure of cells to chemical stressors, namely, NaAsO2 and CdCl2, also enhanced the dissociation of L-hsp27. hsp27 that had been labeled with [32P]H3PO4 in U251 MG cells was detected mostly in fractions that contained S-hsp27, and the incorporation of radioactivity to S-hsp27 was enhanced under conditions that stimulated the dissociation of L-hsp27. L-hsp27 present in the (NH4)2SO4 fraction (0-50% saturation) of cell extracts were dissociated to 32P-labeled hsp27 when incubated in the presence of [gamma-32P]ATP and Mg2+. These results indicate that the molecular configuration of hsp27 in cells is determined in part by phosphorylation and dephosphorylation of this protein by protein kinase(s) and phosphatase(s) and, moreover, that the rapid dissociation of the aggregated form of hsp27 by phosphorylation might be involved in a cellular defense mechanism for protection against stress.

Radioiodination of a monoclonal antibody using N-succinimidyl 5-iodo-3-pyridinecarboxylate

The potential utility of N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) for the radioiodination of monoclonal antibodies was investigated. Paired-label studies were performed using the anti-tenascin antibody 81C6 in athymic mice bearing subcutaneous D-54 MG human glioma xenografts. Radiolabeling was also done using N-succinimidyl 3-iodobenzoate (SIB). Radioiodination of SIPC and SIB both proceeded in 60–80% yield, but protein coupling efficiencies with SIB were higher (76 ± 16 vs 60 ± 7%). Immunoreactivity and affinity of both preparations were similar. Using SIPC, thyroid uptake was quite low, decreasing from 0.3% at day 1 to 0.05% at day 8. Tumor uptake reached 46 ± 11% injected dose/g at day 1 but declined gradually thereafter. This apparent decline reflected the rapid growth of these xenografts since tumor accumulation expressed as percentage of injected dose remained nearly constant up to day 9. These results suggest that SIPC, like SIB, offers significant advantages for labeling antibodies when compared with conventional protein iodination methods.

Responses to heat shock of αB crystallin and HSP28 in U373 MG human glioma cells

Responses to heat shock of αB crystallin and small heat-shock protein HSP28, which are expressed at considerable levels in human astroglioma U373 MG cells (2–4 μg of each per mg soluble protein in confluent cultures), were analysed quantitatively by specific immunoassays. Concentrations of αB crystallin and HSP28 in soluble extracts of U373 MG cells decreased to about 50% of original values, with an increase in the insoluble fraction, during heat treatment for 15 min at 45°C. The concentrations of αB crystallin and HSP28 increased gradually upon return to 37°C, reaching and then exceeding the control levels within 5 h and 10 h, respectively, after heat shock. During centrifugation on sucrose density gradients both αB crystallin and HSP28 in extracts from untreated and heat-treated cells sedimented at the same position, which corresponded to a molecular mass of > 540 kDa . This result suggests that the sizes of aggregates of the two proteins in the cytoplasm are not affected by heat shock. Both αB crystallin and HSP28 in an extract of U373 MG cells were trapped on and coeluted from an affinity column prepared with antibodies against αB crystallin. These results suggest that the two proteins are also associated in U373 MG cells.

Antiviral activity of FIAU versus strains of CMV sensitive and resistant to ganciclovir

To circumvent the in vivo instability of 5-iodo-2′-deoxyuridine (IUdR), a 2′-fluorine-substituted analogue, 5-iodo-1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (FIAU) recently has been introduced. To facilitate the preparation of radioiodinated FIAU as well as its astatinated analogue, a tin precursor, 5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (FTAU) was synthesized. Both [125/131I]FIAU and 5-[211At]astato-1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (FAAU) were prepared from FTAU in more than 85% radiochemical yield under mild conditions. The in vitro serum stability of both fluorine-substituted derivatives was higher than that of the corresponding unsubstituted parents. The enhanced stability of fluorinated derivatives was even more apparent in whole blood. The uptake of [125I]FIAU in D-247 MG human glioma cells in vitro was 20-fold higher than that of [125I]IUdR over an activity concentration range of 5–100 kBq/mL; the uptake of FAAU was not significantly different from that of 5-[211At]astato-2′-deoxyuridine (AUdR). Accumulation of radioiodine in mouse thyroid in vivo with [131I]FIAU was fivefold lower than [125I]IUdR, indicating that the former was less susceptible to deiodination. The tissue uptake of FAAU was similar to that reported for AUdR.

L-Buthionine-Sulfoximine-Mediated Radiosensitization in Experimental Interstitial Radiotherapy of Intracerebral D-54 MG Glioma Xenografts in Athymic Mice

Abstract An intracranial (i.c.) interstitial radiotherapy model in athymic nude mice bearing i.c. D-54 MG human glioma xenografts was developed, allowing evaluation of the therapeutic benefits seen after L-buthionine-S, R-sulfoximine (L-BSO)-mediated depletion of tumor glutathione levels. Administration of L-BSO [2.5 mmol/kg intraperitoneal injections x 4 doses plus concomitant availability in acidified (pH 3.0) drinking water at a concentration of 20 mM] resulted in depletion of tumor glutathione levels to 0.15 mmol/g wet weight (7.9% of control). The therapeutic activity of i.c. interstitial radiotherapy with an 125I seed was enhanced after L-BSO-mediated glutathione depletion, with increases in median survival of 13.4 to 30.5% over that seen with 125I seeds alone. These studies demonstrate a potential role for BSO in enhancing the therapeutic activity of interstitial radiotherapy.

L-buthionine-sulfoximine-mediated radiosensitization in experimental interstitial radiotherapy of intracerebral D-54 MG glioma xenografts in athymic mice

An intracranial (i.c.) interstitial radiotherapy model in athymic nude mice bearing i.c. D-54 MG human glioma xenografts was developed, allowing evaluation of the therapeutic benefits seen after L-buthionine-S,R-sulfoximine (L-BSO)-mediated depletion of tumor glutathione levels. Administration of L-BSO [2.5 mmol/kg intraperitoneal injections x 4 doses plus concomitant availability in acidified (pH 3.0) drinking water at a concentration of 20 mM] resulted in depletion of tumor glutathione levels to 0.15 mumol/g wet weight (7.9% of control). The therapeutic activity of i.c. interstitial radiotherapy with an 125I seed was enhanced after L-BSO-mediated glutathione depletion, with increases in median survival of 13.4 to 30.5% over that seen with 125I seeds alone. These studies demonstrate a potential role for BSO in enhancing the therapeutic activity of interstitial radiotherapy.

Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.

alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model.

Use of cytofluorometry to evaluate binding of antibodies to the cytoskeleton of cultured cells.

Immunocytochemistry is routinely used to examine the occurrence and distribution of cytoskeletal proteins in cells, but the results are usually evaluated visually and subjectively. Little use has been made of the potential the method offers for quantitative work. Here we report on application of cytofluorometry to quantify binding of antibodies to the cytoskeleton of U-251 MG human malignant glioma cells in culture. The results show that cytofluorometry is a simple and reliable procedure for: (a) determining the optimal concentrations of primary and secondary antibodies and other labeling reagents; (b) evaluating the binding specificity of commercial secondary antisera; and (c) evaluating the effect of different preparatory procedures on preservation of and binding of antibodies to cytoskeletal structures. Experiments with a monoclonal antibody to tubulin show that preservation of tubulin is very sensitive to the preparatory procedures used. Maximum labeling of tubulin in intact cells was obtained when the cells were pre-fixed with formaldehyde before permeabilization with solvent. Maximum labeling of tubulin in Triton-extracted cytoskeletons was achieved by pre-fixing the cells with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate), extracting with Triton in a microtubule-stabilizing buffer, and post-fixing with formaldehyde. GTP was not required to preserve tubulin in cytoskeletons.