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Malignant gliomas are highly proliferative and invasive tumors with poor prognosis. We investigated the influence of Interferon-gamma (IFN-gamma) on the human malignant glioma cell line A172, measuring cell viability (MTT-test), proliferation (3H-thymidine-uptake), cell death (FACS) adhesion to hyaluronic acid (HA, adhesion-assay) and migration (Boyden-chamber). IFN-gamma significantly decreased cell viability and proliferation. Measured by FACS, an up-regulation of CD95 expression has been shown in combination with an increased rate of cell death, first seen after 96 hours IFN-gamma treatment. Adhesion to HA was decreased after pre-treatment with IFN-gamma. This was not mediated by down-regulation of the main HA-receptor CD44, since IFN-gamma did not change CD44 expression. IFN-gamma-treated cells showed a significantly diminished migration rate through a native or HA-coated 8-microm polycarbonate membrane. To summarise, IFN-gamma influences both the main characteristics of malignancy: it decreases cell proliferation and induces cell death, further it diminishes migration of A172 human glioblastoma cells.

The cytokine interleukin (IL)-1 is known to be involved in angiogenesis and metastasis. A prerequisite for IL-1 signalling is the presence of its receptor. Previously we have shown that glioblastoma cells express the IL-1 receptor type I (IL-1RI). In this study we analysed 11 breast tumour specimens for IL-1RI expression using the reverse transcriptase (RT) polymerase chain reaction (PCR). We found all the 11 breast tumours were positive for IL-1RI. This suggests that paracrine or autocrine produced IL-1 mediated signalling via IL-IRI might take place in breast tumours to control the production of pro-tumourigenic factors such as angiogenic factors and support further progression of tumour growth and metastasis.

Valproic acid (VPA) has been considered as a possible treatment agent for malignant gliomas. In order to characterise the possibilities of VPA, we investigated the effects on cell migration and proliferation. Human cell lines T98G, A172, 85HG66 and 86HG39 were treated with VPA or left untreated, afterwards Boyden chamber assay was used for measuring vertical migration. In a second assay cells were stimulated to create spheroids and spheroid migration was measured. Proliferation was assessed using a cell counter. VPA decreased proliferation of 86HG39 > A172 > 85HG66 cells, whereas T98G remained uninfluenced. The influence of VPA on migration was different; whereas VPA dose-dependently stimulated migration of 86HG39 cells, migration of T98G and 85HG66 decreased, whereas A172 cells remained uninfluenced. Only 86HG39 and A172 cells created spheroids. In both cell lines Boyden-chamber-findings were confirmed by analysing the influence of VPA on spheroid migration. These non-uniform data demonstrate that the benefit of VPA in glioma treatment is not clear and needs further investigation.

Because of the outstanding importance of the glucocorticoid Dexamethasone (DEX) as supportive therapy in the management of brain tumours, the direct effect of DEX on tumour cell proliferation is of particular interest. Previous in vitro studies led to contradictory results. To characterise more precisely the influence of DEX, we investigated the glioblastoma multiforme (GM) cell lines A172, T98G and 86HG39. Cells were treated with DEX concentrations ranging from 5 x 10(-9) to 5 x 10(-5) M from 24 to 240h under different treatment conditions. Influence of DEX on glioma cell viability was assessed daily for 5 days by MTT-assay: (I) with continuous DEX incubation (acute treatment), (II) in a recultivation period without DEX after 5 days of DEX pre-incubation (pre-treatment), (III) with continuous DEX incubation after 5 days of DEX pre-incubation (combination treatment). DEX acute treatment led to strongly decreased proliferation of A172 cells, whereas T98G and 86HG39 cells remained uninfluenced. In opposite, a time-delayed inhibition of cell proliferation was observed in all three cell lines after DEX pre-treatment. Combination treatment induced a significant increase of the inhibitory effect in A172 and T98G cells. These data show a variable, partial time-dependent inhibitory effect of DEX on the proliferation of GM cells and may open new treatment strategies for malignant brain tumours.

Malignant gliomas are frequent and the prognosis is poor. The cytokine interferon gamma (IFN-gamma) enhances several immune phenomena and may be used in immunotherapy of tumours. Therefore we investigated the influence of IFN-gamma on human cell lines T98G, U87MG, 86HG39 and 85HG66, measuring cell viability (MTT-test) and proliferation (3H-thymidine uptake). IFN-gamma markedly decreased viability and proliferation of all investigated cell lines. Expression of CD44 and adhesion to hyaluronic acid (HA) are involved in glioma invasion. Influence of IFN-gamma on these two features has also been investigated. IFN-gamma markedly decreased HA-adhesion in all three investigated cell lines, whereas CD44 expression remained uninfluenced. To summarise, IFN-gamma strongly decreased cell growth and HA-adhesion of malignant glioma cell lines in vitro. We suggest further investigations to characterise better the role of IFN-gamma as a treatment opportunity for malignant gliomas.

We have previously demonstrated that treatment of wild-type (wt) T98G malignant glioma cells with Cisplatin (CDDP) led to a resistant phenotype. It has been demonstrated that interleukin 1 (IL-1) potentiates the cytotoxic effect of CDDP and that IL-6 decreases cytotoxicity by inhibition of apoptosis in cancer cells. Here we examined the influence of IL-1 and IL-6 on the sensitivity of resistant and wt T98G cells. Using semi-quantitative PCR reactions in three independent experiments, resistant glioma cells revealed a decreased IL-1alpha (50.3+/-7.2), IL-1beta (56.0+/-4.0) and IL-6 (44. 3+/-18.2) mRNA content compared to wt cells (100%;P<0.05). Resistant and wt cells were positive for the receptors IL-1RI and IL-6R (PCR). To investigate whether IL-1alpha, IL-1beta or IL-6 changes the sensitivity of the resistant and wt cells towards CDDP, cells were incubated up to 7 days with 10(-5) M CDDP and with different concentrations (0, 0.01, 0.1, 1 ng/ml) of cytokine. Sensitivity was tested in a colorimetric assay (MTT). IL-6 did not influence the sensitivity towards CDDP of either wt or resistant cells, while IL-1alpha and IL-1beta enhanced sensitivity of resistant cells to CDDP. These data suggest that autocrine IL-1 production is involved in the mechanisms of resistance in T98G cells., (Copyright 1999 Academic Press.)

Despite surgery and adjuvant cytotoxic therapy anaplastic astrocytoma, glioblastoma and diffuse intrinsic brain stem glioma continue to have dismal prognosis. Differentiation induction is a new approach taking into account that malignant glioma cells share many features with immature glial progenitor cells that are capable of terminal differentiation. The concept of differentiation therapy is currently evaluated for several pediatric malignancies with or without multimodal standard therapy. Valproic acid (VPA) is a branched chain fatty acid that is able to inhibit proliferation of neuroectodermal cells and to induce these cells along neuronal or glial lineage. Preclinical studies have shown that VPA inhibits growth of human and rodent glial tumor cells in vitro and induces a distinct mature glial phenotype. In addition, growth of human neuroblastoma cells is inhibited in vitro and in vivo and exhibits marked evidence of differentiation. Treatment of neuroblastoma and glioma cells with VPA was accompanied by changes of surface molecule expression that enhance immunogenicity and reduce their capability to metastasize. The antitumoral effects observed in preclinical studies were reached at concentrations that are readily achieved in patients treated with VPA for epilepsy. Epilepsy patients receiving VPA have significantly enhanced hemoglobin F levels, supporting the hypothesis that nontoxic levels of VPA can induce cellular differentiation. Broad clinical experience with VPA and its low toxicity encourage the evaluation of VPA in patients that have been submitted to postoperative combined chemo- and radiotherapy for pediatric malignant glioma.

Malignant gliomas are often treated with cisplatin (cis-diamminedichloroplatinum(II), CDDP) and radiation but results remain unsatisfactory. The development of resistance might explain the poor prognosis. The aim of this study was to investigate whether two human malignant glioma cell lines, 86HG39 and A172, develop resistance to CDDP and/or radiation after CDDP pretreatment. The cells are incubated three times with 10(-6) M CDDP for 24 h, allowed to recover from the treatment and then tested for sensitivity to CDDP and radiation (9 Gy, 60Co) using a colorimetric assay (MTT). A172 pretreated and wild-type cells showed no difference in sensitivity to CDDP, whilst 86HG39 cells became more sensitive following pretreatment. This indicates that no resistant phenotype towards CDDP developed in any of the cell lines. In contrast, the CDDP-pretreated cells, after radiation, had significantly higher growth rates compared with the wild-type cells in both cell lines (A172: 4.4 +/- 0.5 versus 2.5 +/- 0.3, respectively, 192 h after radiation; 86HG39: 6.2 +/- 0.7 versus 2.3 +/- 0.3, respectively, 216 h after radiation; P < 0.05) indicating resistance against radiation. The level of glutathione (GSH), which is known to mediate resistance against radiation, was 157.2 +/- 61.3 ng/mg protein in A172 cells and 93.2 +/- 16.9 ng/mg protein in 86HG39 cells, and there was no significant difference between levels in wild-type and pretreated cells (A172: 130.1 +/- 34; 86HG39: 81.6 +/- 10.4). These data show that CDDP pretreatment can induce resistance against radiation in vitro independently of CDDP cross-resistance mediated by a mechanism different from GSH.

The mechanisms leading to rapid invasive growth of malignant gliomas are poorly understood. Expression of the hyaluronic acid (HA) receptor CD44 and adhesion to HA are involved in invasive properties. Our previous studies have shown that malignant glioma cells are able to adhere to extracellular HA. Here we investigated expression of the hyaluronic acid receptor CD44 protein in five human (T98G, A172, U87MG, 86HG39, 85HG66) and two rat (C6, 9L) glioma cell lines. Influence of anti-CD44 antibody and hyaluronidase-preincubation on the HA-binding was determined using HA/BSA (bovine serum albumin)-coated culture plates. While all gliomas were highly positive for CD44 with no differences in the number of positive staining cells, median fluorescence intensity decreased as follows: C6>T98G>9L>85HG66> 86HG39>A172>U87MG. Using HA/BSA coated culture plates the relative levels of specific adhesion to HA were determined as T98G>A172>9L>86HG39>U87MG> 85HG66. C6 cells failed to bind HA specifically. Incubation with anti-human-CD44 MAb significantly decreased HA-adhesion of T98G, A172, 85HG66 and U87MG human glioma cells. However the binding capacity was completely blocked only in 85HG66 cells. The three other cell lines kept a specific HA-adhesion after saturation of the receptor. Hyaluronidase pretreatment markedly enhanced HA-adhesion of C6 and 9L rat glioma cells. These results suggest that (i) HA-adhesion of malignant glioma cells is mainly, but not only, mediated by CD44, (ii) expression of CD44 does not correspond with adhesion capacity and (iii) cell-bound glycosaminoglycans may influence glioma cell adhesion to extracellular HA.

Oxazaphosphorines are inactive anticancer prodrugs that are bioactivated by hepatic cytochrome P450. Besides hepatic metabolism, there is increasing interest in the possibility of intratumoral activation of oxazaphosphorines by P450. Therefore, we investigated the expression of P450 (CYP3A4, CYP3A5, CYP2C9) by RT-PCR in 10 different brain tumor samples. Because P450 may be downregulated by interleukin-1 (IL-1) and IL-6, the receptors for IL-1 and IL-6 were analyzed. None of the brain tumors was positive for CYP3A4 whereas CYP3A5 was detected in 3 out of 10 tumors (two meningeomas, one medulloblastoma grade IV). All five gliomas, an ependymoma, and a lymphoma-metastase gave no signal. CYP2C9 mRNA was present in every sample studied. All samples were positive for IL-1 and IL-6 receptors. In summary, we have demonstrated that tumors of the CNS express P450, indicating that activation of prodrugs like oxazaphosphorines may take place intratumorally. However, the most abundantly hepatically expressed CYP3A4 enzyme is absent in the brain tumor samples. The presence of the IL-1 and IL-6 receptors opens the possibility that the wellknown downregulating influence of these cytokines also takes place in brain tumors.

Recently we were able to show that valproic acid (VPA) induces growth-arrest and differentiation of human neuroblastoma cells. Hence we investigated in vitro the antitumoral effects of VPA on malignant gliomas by determining cell proliferation and expression of CD56 and CD44 of human T98G, A172, 85HG66, 86HG39 and rat C6 cell lines. VPA at concentrations ranging from 0.1 to 1 mM strongly inhibited proliferation of A172, 86HG39, 85HG66 and C6 cells in a dose-dependent manner, whereas T98G cell growth remained unchanged. All human glioma cells were highly positive for CD44, whereas CD56 was differently expressed. After 7 days of incubation with 1mM VPA CD56 expression was markedly increased in T98G, A172 and 85HG66 cells, whereas CD44 expression was decreased in all human cell lines. These data suggest that VPA has antitumoral effects on malignant glioma cells. Therefore we consider VPA as a potent therapeutic agent for treatment of these tumors.

The mechanisms underlying the rapid invasive growth of malignant gliomas are poorly understood. Adhesion to extracellular hyaluronic acid (HA) has been implicated in the invasive properties of tumor cells. We investigated the HA binding capacity of human (T98G, A172, U87MG, 86HG39, 85HG66) and rat (C6, 9L) glioma cell lines by means of HA coated, bovine serum albumin (BSA)-blocked (HA/BSA) and only BSA-blocked culture plates. Results were compared with adhesion to native wells (100% adhesion). Adhesion to HA/BSA was high for T98G (84.4%), medium for 86HG39 (36%), 9L (33.1%), A172 (35.5%) and low for 85HG66 (21.3%) and U87MG (26.8%). Adhesion to only BSA-coated wells was significantly lower in all these cell lines, suggesting a specific HA-adhesion. Only C6 showed similar adhesion to HA/BSA and BSA alone, therefore, C6 failed to bind HA specifically. These results suggest that adhesion to extracellular HA might be involved in the invasion of some gliomas.

Background: The prognosis of children with malignant gliomas is poor. A new therapeutic strategy using interferon-gamma (IFN-g) as an immunostimulator substance will start in Germany in 1997. One of the therapeutic problems in malignant gliomas is the high invasiveness of the tumor cells. Adhesion to the extracellular matrix molecule hyaluronic acid (HA) is involved in invasion. We addressed the question of whether IFN-g (i) influences the proliferation and (ii) changes the HA-binding capacity of malignant glioma cells., Method: T98G glioblastoma cells were incubated up to 4 days with IFN-g (30 and 300 IE/ml). Proliferation was measured by a colorimetric assay (MTT) and compared with untreated controls. Expression of the HA-receptor CD44 was determined by FACS-analysis using the mouse monoclonal antibody J-173. HA-adhesion was investigated using HA-coated (3 mg/ml), bovine serum albumin blocked 24-well-plates and compared with uncoated wells., Results: FN-g (300 IE/ml) inhibited proliferation to 76.3% (p < 0.0001) after 48 hours compared with untreated controls. This effect was mediated not only by inhibition of proliferation, but also by induction of cell-death, first seen 72 h after IFN-g incubation. 24 hours later only 24% of treated cells survived. 93.1% of T98G cells expressed CD44 (FACS-analysis). A specific HA-adhesion of glioma cells was shown: 85.5% of the cells adhered to HA, 13.3% to BSA compared with controls. 300 IE/ml IFN-g decreased HA-adhesion significantly (p < 0.001) to 17%, whereas BSA-adhesion remained unchanged., Conclusion: IFN-g inhibits tumor cell proliferation and diminishes the invasive properties of glioma cells via reduction of HA binding capacity. Our results support the use of IFN-g in the therapy of malignant gliomas.

Malignant gliomas are relatively resistant to radiation and chemotherapy. To investigate whether cisplatin (cis-diamminedichloroplatinum(II), CDDP) causes resistance we pretreated C6 cells with 10(-6) M CDDP for 24 hours and then tested their sensitivity in a colorimetric assay. Pretreated cells developed resistance to CDDP (resistance factor 2.0) and radiation (survival after 9 Gy 60Co: 36.4% +/- 5.5 versus 28.6% +/- 5.2, p = 0.005). Glutathione levels of pretreated cells were higher (51.7 +/- 13.8 ng/mg protein) than in wt cells (40.4 +/- 13.2, p = 0.029). Addressing the mechanisms we established 4 wild type subclones with different CDDP sensitivities. However, cross-resistance to CDDP (survival: 60.7% +/- 3.5 versus 7.2% +/- 0.5, respectively p = < 0.001) and radiation (29.7% +/- 2.6 versus 12.9% +/- 0.8, p = < 0.001) could also be induced in a subclone showing involvement of mutation. These data suggest that CDDP can induce resistance mediated via induced mutation and increased GSH levels.

Malignant gliomas are often treated with cisplatin (cis-diamminedichloroplatinum(II), CDDP) and radiation but results remain unsatisfactory. To investigate whether CDDP induces radioresistance in glioma, T98G human glioblastoma cells were pretreated 5 times with 10(-6)M CDDP for 24 hours and then the sensitivity of wild type (wt) and pretreated cells towards radiation (9Gy 60Co) and CDDP was tested in a colorimetric assay (MIT). The growth rates of wt and pretreated cells were 1.8 +/-0.2 and 3.1 +/- 0.2 respectively (p = 0.000155) 216 hours post radiation. Pretreated cells also developed resistance to CDDP (resistance factor 2.35). Glutathione (GSH) which potentially mediates resistance to both treatments was measured. Incubation for 6 hours with 10(-5) M CDDP increased GSH levels by a factor of 2.28 (p < 0.0001). However, neither basal nor increased levels differed between wt and pretreated cells. These data show that CDDP pretreatment can induce resistance against radiation and CDDP independently of GSH.