Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Enhanced survival of glioma bearing rats following boron neutron capture therapy with blood-brain barrier disruption and intracarotid injection of boronophenylalanine

Boronophenylalanine (BPA) has been used for boron neutron capture therapy (BNCT) of brain tumors in both experimental animals and humans. The purpose of the present study was to determine if the efficacy of BNCT could be enhanced by means of intracarotid (i.c.) injection of BPA with or without blood-brain barrier disruption (BBB-D) and neutron irradiation using a rat brain tumor model. For biodistribution studies, F98 glioma cells were implanted stereotactically into the brains of Fischer rats, and12 days later BBB-D was carried out by i.c. infusion of 25% mannitol (1.373 mOsmol/ml), followed immediately by i.c. administration of 300, 500 or 800 mg of BPA/kg body weight (b.w.). At the 500 mg dose a fourfold increase in tumor boron concentration (94.5 g/g) was seen at 2.5 hours after BBB-D, compared to 20.8 g/g in i.v. injected animals. The best composite tumor to normal tissue ratios were observed at 2.5 hours after BBB-D, at which time the tumor: blood (T: Bl) ratio was10.9, and the tumor: brain (T: Br) ratio was 7.5, compared to 3.2 and 5.0 respectively for i.v. injected rats. In contrast, animals that had received i.c. BPA without BBB-D had T: Bl and T: Br ratios of 8.5 and 5.9, respectively, and the tumor boron concentration was 42.7g/g. For therapy experiments, initiated 14 days after intracerebral implantation of F98 glioma cells, 500 mg/kg b.w. of BPA were administered i.v. or i.c. with or without BBB-D, and the animals were irradiated 2.5 hourslater at the Brookhaven Medical Research Reactor with a collimated beam of thermal neutrons delivered to the head. The mean survival time for untreated control rats was 24 ± 3 days, 30 ± 2 days for irradiated controls, 37 ± 3 days for those receiving i.v. BPA, 52 ± 15 days for rats receiving i.c. BPA without BBB-D, and 95 ± 95 days for BBB-D followed by i.c. BPA and BNCT. The latter group had a 246% increase in life span (ILS) compared to untreated controls and a 124% ILS compared to that of i.v. injected animals. These survival data are the best ever obtained with the F98 glioma model and suggest that i.c. administration of BPA with or without BBB-D may be useful as a means to increase the efficacy of BNCT.     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.     

Clinical application of18F-FUdR in glioma patients — PET study of nucleic acid metabolism

Summary Positron emission tomography was used to investigate the metabolism of nucleic acids by¹⁸F-fluoro-2-deoxyuridine (¹⁸F-FUdR) in 22 patients with gliomas. Sixteen cases of high grade glioma clearly demonstrated a region of high activity with a differential absorption rate (DAR) of 0.64 ± 0.34. Six cases of low grade glioma failed to reveal a positive image of the tumor and the DAR in tumor was 0.21 ± 0.042 (p < 0.01). This PET-¹⁸F-FUdR study succeeded in differentiating high and low grade gliomas from the view point of nucleic acid metabolism.     

反义胰岛素样生长因子—1mRNA治疗大鼠脑胶质瘤模型的动态MR研究

目的：动态观察大鼠Ｃ６胶质瘤模型的自然生长过程及反义ＩＧＦ－１ｍＲＮＡ对胶质瘤的治疗效果和治疗机制。方法：将３２只Ｗｉｓｔａｒ大鼠分成４组,每组８只,（１）对照组（Ｃ组）于右尾状核接种Ｃ６鼠胶质瘤细胞。（２）治疗组（Ｔ组）：于右尾状核接种Ｃ６细胞后的第７、１０天在接种点注射脂质体包裹的质粒Ｐ－ａｎｔｉ－ＩＧＦ－１,对胶质瘤进行治疗于右尾状核接种经反义ＩＧＦ－１ｍＲＮＡ转染的Ｃ６胶质瘤细胞。（４）Ｋ     

Expression of the extraneuronal monoamine transporter (uptake2) in human glioma cells

Tritiated methylphenylpyridinium ([³H]MPP⁺), a substrate of the neuronal and extraneuronal noradrenaline transporter (uptake₁ and uptake₂, respectively) and of the organic cation transporter (OCT1), was used to characterize the amine transport system of the established human glioma cell line SK-MG-1.Uptake of [³H]MPP⁺ (25 nM) into SK-MG-1 cells increased linearly with time for up to 15 min. Selective uptake₁ inhibitors (e.g. (+)oxaprotiline) or omission of Na⁺ or Cl^– ions did not affect [³H]MPP⁺ uptake, whereas uptake₂ inhibitors such as O-methyl-isoprenaline (OMI) or corticosterone as well as depolarizing concentrations of K⁺ or Ba²⁺ strongly reduced [³H]MPP⁺ uptake. Initial rates of OMI(100 M)-sensitive [³H]MPP⁺ uptake were saturable, with a K_m of about 17 M and a maximal rate of about 50 pmol/ (min × mg protein). IC₅₀ (or K_i) values for inhibition of [³H]MPP⁺ uptake by substrates and inhibitors of uptake₂ or OCTI were highly significantly correlated with published IC₅₀ values for inhibition of uptake₂ but not with corresponding values for inhibition of OCT1.The results presented here clearly demonstrate that human glioma cells express an uptake₂ transporter. Thus, glial cells in the human central nervous system endowed with this transporter are likely to contribute to the inactivation of neuronally released noradrenaline.     

Interferon-β inhibits proliferation and progression through S phase of the cell cycle in five glioma cell lines

Summary The growth inhibitory effect of IFN- was evaluated in 5 human glioma cell lines (AO2V4, GJC, GJR, NN and NNR) and in normal astrocyte cultures (SC and TM). All 5 glioma cell lines showed an anti-proliferative response to IFN- whereas normal glial cells were non-responsive. IFN- at 10, 100 and 500 U/ml lead to a 30%,70% and 80% relative decrease in cell number after 12 days, respectively in AO2V4 cells. GJC and GJR cell lines also responded significantly to the lowest concentration of IFN- tested and at 500 U/ml the relative cell number decreased 55%. The NN and NNR cells were the least responsive to IFN- with maximum growth inhibition of 30% at 500 U IFN-/ml. Following treatment with IFN-, AO2V4, GJC, GJR and normal astrocytes all expressed mRNA encoding the anti-viral protein, 2-5A synthetase demonstrating that IFN- bound to receptors on all four cell lines and activated signal transduction pathways required for induction of an anti-viral protein. A determination of the relative number of viable cells showed that none of these cells exhibited a significant decrease in cell viability. Since the antiproliferative response to IFN- was not primarily due to cell death, the effect of IFN- on cell cycle progression was evaluated by flow cytometry. All treated glioma cell lines showed a relative increase in proportion of cells in S phase. AO2V4 cells had a 50%–80% increase in the percentage of cells in S phase, whereas GJC, GJR and NNR had percentage increases of 20%–40%. IFN- treatment of normal astrocytes did not significantly alter their cell cycle profile. These data suggest that IFN- exerts its antiproliferative effect on glioma cells by arresting the ordered progression through S phase or decreasing entry into G₂/M phase of the cell cycle.     

Cerebellar astrocytomas in children

Cerebellar astrocytomas, as a group, carry a more favorable prognosis than most other brain tumors, because these neoplasms generally are histologically benign and amenable to extensive resection. However, it is clear that a number of factors have an impact on prognosis. In particular, resection extent has been strongly associated with progression-free survival: patients undergoing gross total resection appear to have a substantially better prognosis than those undergoing incomplete resection. Brainstem invasion, which is the factor that most often precludes a complete resection, has also been associated with a less favorable prognosis. In addition, histological features indicative of malignancy are clearly associated with a poor outcome.In contrast to the above observations, which have been established convincingly in the literature, a number of issues regarding cerebellar astrocytomas remain unresolved. First, the correlation between histology and prognosis among patients with low-grade cerebellar astrocytomas is uncertain: in some series, pilocytic astrocytomas have been associated with a better prognosis than non-pilocytic tumors, but in other studies, no such relationship has been observed. Second, the role of radiotherapy after incomplete resection of a low-grade cerebellar astrocytoma remains problematic. In view of the lack of convincing data in this regard, many groups, including our own, defer radiotherapy until there is evidence of progressive disease that is surgically unresectable. Finally, the frequency of follow-up in patients with cerebellar astrocytomas remains largely empirical. Although most recurrences are detected within a few years after initial surgery, late recurrences are well known, which raises the question of when and if such patients should be regarded as cured of their disease. Long-term multi-institutional natural history studies are in progress to address the above issues.     

Neuropathological and neurophysiological effects of interstitial white matter autologous and non-autologous protein containing solutions: Further evidence for a glioma derived permeability factor

Summary The feline infusion model of brain edema was used to evaluate the pathophysiological effects of 0.6ml infusions of autologous serum protein (66%), human serum protein (66%), human glioma cyst fluid and a tissue culture medium (TCM) on the structure and function of the forebrain white matter. These infusions increased local white matter water content by between 10.8 and 12.5 ml/100 g brain and were associated with moderate increases in ICP and CSF outflow resistance and a significant decrease in lumped craniospinal compliance. Cortical somatosensory potentials, motor evoked potentials, EEG and local cerebral blood flow (rCBF) at normocapnia were generally unchanged by the various infusions. All infusates except the 66% autologous serum protein infusion impaired rCBF CO₂ reactivity. Histologically all infusates caused marked extracellular edema. The autologous serum protein infusion caused no additional histological changes whereas the glioma cyst infusates caused profound endothelial and astrocytic swelling, focal endothelial necrosis, basement membrane disruption, perivascular microglial reaction and pavementation and perivascular migration of polymor-phonuclear leukocytes. Similar but less marked changes were seen after infusion of human serum protein whilst the TCM produced only minimal changes. The intensity and extent of Evans Blue extravasation into the forebrain white matter was greatest with glioma cyst infusates and with all infusions reflected the extent to microvascular changes.These studies show that products derived from gliomas cause additional damage to the blood-brain-barrier than that caused by non-autologous serum proteins. These results add further support for the existence of glioma derived permeability factors (GDPF), but suggest neither serum proteins nor glioma derived compounds in the white matter interstitium significantly influence local electrophysiological function. Some limitations of the infusion edema model when using non-autologous infusions and difficulties quantitating brain dysfunction are emphasised.Preliminary results had been presented at the symposium on Brain Edema VIII, which took place at Bern, Switzerland, in June 1990 and have been published in: Reulenet al (eds) 1990. Brain Edema VIII, Acta Neurochirurgica (Wien) [Suppl] 51: 71–73