T9 Glioma Cells Research Articles

Molecular Mechanisms of Paraptosis Induction: Implications for a Non-Genetically Modified Tumor Vaccine

Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Previously, rodent and human monocytes/macrophages killed glioma cells bearing the membrane macrophage colony stimulating factor (mM-CSF) through paraptosis, but the molecular mechanism of this killing process was never identified. We have demonstrated that paraptosis of rat T9 glioma cells can be initiated through a large potassium channel (BK)-dependent process initiated by reactive oxygen species. Macrophage mediated cytotoxicity upon the mM-CSF expressing T9-C2 cells was not prevented by the addition of the caspase inhibitor, zVAD-fmk. By a combination of fluorescent confocal and electron microscopy, flow cytometry, electrophysiology, pharmacology, and genetic knock-down approaches, we demonstrated that these ion channels control cellular swelling and vacuolization of rat T9 glioma cells. Cell lysis is preceded by a depletion of intracellular ATP. Six-hour exposure to BK channel activation caused T9 cells to over express heat shock proteins (Hsp 60, 70, 90 and gp96). This same treatment forced HMGB1 translocation from the nuclear region to the periphery. These last molecules are “danger signals” that can stimulate immune responses. Similar inductions of mitochondrial swelling and increased Hsp70 and 90 expressions by BK channel activation were observed with the non-immunogenic F98 glioma cells. Rats injected with T9 cells which were killed by prolonged BK channel activation developed immunity against the T9 cells, while the injection of x-irradiated apoptotic T9 cells failed to produce the vaccinating effect. These results are the first to show that glioma cellular death induced by prolonged BK channel activation improves tumor immunogenicity; this treatment reproduces the vaccinating effects of mM-CSF transduced cells. Elucidation of strategies as described in this study may prove quite valuable in the development of clinical immunotherapy against cancer.

Human Allogeneic and Murine Xenogeneic Dendritic Cells Are Cytotoxic to Human Tumor Cells via Two Distinct Pathways

Human monocyte-derived dendritic cells (DCs), stimulated with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 1 week, major histocompatibility complex killed human tumor cells in 24-hour cytotoxicity assays. These immature DCs were >90% CD11c, major histocompatibility complex class II(+), but <1% were CD83(+) cells. Within 24 hours, these DCs ingested tumor membranes. The DC cells also lysed Jurkat lymphoma cells, but not Jurkat cells genetically knocked out of the Fas-associated death domain (FADD) or caspase-8. DC2.4, a cloned murine DC line, also displayed cytotoxicity toward U-251 cells, although these murine DCs were less potent than human DC. DC2.4 did not kill Jurkat cells, rat T9 glioma cells, or human Caco-2 colon cancer cells, suggesting that a unique receptor or ligand interaction exists between the DC and U-251 cells. This interaction was destroyed by the paraformaldehyde fixation of the tumor cells. Supernatants from the cultures of DC2.4 and tumor cells were analyzed by the Griess reaction for signs of nitric oxide (NO) production. Augmented NO production occurred in DC2.4/U-251 and DC2.4/Jurkat cultures but was not seen in the human DC/U-251 cultures. These studies suggest that DCs possess different mechanisms of tumoricidal activity.

A Bex‐cycle built for two

Neurotrophins promote neuronal survival, but it has been recognized that they also stimulate differentiation. Indeed, in early studies by Hamburger, Levi‐Montelcini and Cohen, nerve growth factor (NGF) not only promoted survival, but also caused marked neurite outgrowth when added to explants of sensory ganglia (Cohen et al , 1954). The ability of NGF to promote differentiation was particularly well illustrated when Greene & Tischler (1976) showed that a pheochromocytoma cell line (PC12) treated with NGF exited the cell cycle and differentiated into a sympathetic neuron‐like phenotype. Since then, it has been established that the survival and differentiation effects of the neurotrophins are primarily mediated by binding to members of the Trk family of tyrosine kinase receptors (Huang & Reichardt, 2003); however, the first receptor identified for NGF was a 75‐kDa protein referred to as p75 (Chao et al , 1986; Radeke et al , 1987). In the past few years, the p75 receptor has received a lot of attention because of its multifunctional signalling ability. This receptor can promote cell survival or induce apoptosis, regulate neurite outgrowth and promote peripheral myelination, depending on the specific cellular context, the expression of co‐receptors and the activating ligand (Barker, 2004). The role of p75 in promoting differentiation and cell‐cycle arrest has been largely overlooked in recent years, even though this was one of the first cellular responses to be shown to result from the binding of NGF to the receptor. Long considered to be simply a co‐receptor for the Trks, p75 came to be recognized as a bona fide signalling molecule when it was shown to activate sphingomyelinase. This resulted in the production of ceramide, which led to the differentiation of T9 glioma cells as indicated by their cell‐cycle arrest and process outgrowth (Dobrowsky et al , 1994). Although the focus of most subsequent research …

Living T9 glioma cells expressing membrane macrophage colony-stimulating factor produce immediate tumor destruction by polymorphonuclear leukocytes and macrophages via a “paraptosis”-induced pathway that promotes systemic immunity against intracranial T9 gliomas

Cloned T9-C2 glioma cells transfected with membrane macrophage colony-stimulating factor (mM-CSF) never formed subcutaneous tumors when implanted into Fischer rats, whereas control T9 cells did. The T9-C2 cells were completely killed within 1 day through a mechanism that resembled paraptosis. Vacuolization of the T9-C2 cell's mitochondria and endoplasmic reticulum started within 4 hours after implantation. By 24 hours, the dead tumor cells were swollen and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)-positive. Bcl2-transduced T9-C2 cells failed to form tumors in rats. Both T9 and T9-C2 cells produced cytokine-induced neutrophil chemoattractant that recruited the granulocytes into the tumor injection sites, where they interacted with the tumor cells. Freshly isolated macrophages killed the T9-C2 cells in vitro by a mechanism independent of phagocytosis. Nude athymic rats treated with antiasialo GM1 antibody formed T9-C2 tumors, whereas rats treated with a natural killer cell (NK)-specific antibody failed to form tumors. When treated with antipolymorphonuclear leukocyte (anti-PMN) and antimacrophage antibodies, 80% of nude rats formed tumors, whereas only 40% of the rats developed a tumor when a single antibody was used. This suggests that both PMNs and macrophages are involved in the killing of T9-C2 tumor cells. Immunocompetent rats that rejected the living T9-C2 cells were immune to the intracranial rechallenge with T9 cells. No vaccinating effect occurred if the T9-C2 cells were freeze-thawed, x-irradiated, or treated with mitomycin-C prior to injection. Optimal tumor immunization using mM-CSF-transduced T9 cells requires viable tumor cells. In this study optimal tumor immunization occurred when a strong inflammatory response at the injection of the tumor cells was induced.

Irradiated tumor cell vaccine for treatment of an established glioma. II. Expansion of myeloid suppressor cells that promote tumor progression.

These studies report the identification of a population of myeloid suppressor cells (MSC) that are preferentially enriched in the spleens and tumor-infiltrating mononuclear cells (TIMC) from T9.F-vaccinated animals. In this model designed to mimic immunotherapy for an established intracranial (i.c.) glioma, animals were given an i.c. inoculum with 5 x 10(4) T9 glioma cells at day 0, followed by a subcutaneous (s.c.) injection of 5 x 10(6) irradiated T9.F glioma cells 5 days later. Unexpectedly, we found that the survival of these T9.F-vaccinated animals was dramatically shorter than their age-matched counterparts who received only saline injections. Since MSC have previously been demonstrated to be associated with tumor progression, the question arose of whether MSC might play a role in the rapid tumor progression observed in this model. Analysis of the spleen cells and TIMC revealed an increase in the population of myeloid cells expressing granulocytic and monocytic markers. Both the polyclonal and tumor-specific proliferation of splenic T cells and tumor-infiltrating T lymphocytes (T-TIL) from T9.F-vaccinated animals were significantly inhibited in the presence of these myeloid cells. Furthermore, the adoptive transfer of MSC into animals bearing a 5-day T9 glioma caused rapid tumor progression. Reduced survival of the glioma-bearing vaccinated rats was associated with enhanced tumor growth, as well as with an increased density of T-TIL. However, purified T-TIL did not show any discernable signs of inherent defects in terms of their effector functions and T cell receptor (TCR) signal transduction protein levels. Therefore, we believe that an MSC population is responsible for inhibiting the anti-tumor T cell response, resulting in the enhanced growth of the i.c. glioma, and may represent a significant obstacle to immune-based therapies.

T9 glioma cells expressing membrane-macrophage colony stimulating factor produce CD4+ T cell-associated protective immunity against T9 intracranial gliomas and systemic immunity against different syngeneic gliomas

Cloned T9 glioma cells (T9-C2) expressing the membrane form of macrophage colony stimulating factor (mM-CSF) inoculated subcutaneously into rats do not grow and glioma-specific immunity is stimulated. Immunotherapy experiments showed that intracranial T9 tumors present for one to four days could be successfully eradicated by peripheral vaccination with T9-C2 cells. CD4+ and CD8+ T splenocytes from immunized rats, when restimulated in vitro with T9 cells, produced interleukin-2 and -4. Protective immunity against intracranial T9 gliomas could only be adoptively transferred into naive rats by the CD4+ splenocytes obtained from T9-C2 immunized rats. Rats immunized by the T9-C2 tumor cells also resisted two different syngeneic gliomas (RT2 and F98) but allowed a syngeneic NUTU-19 ovarian cancer to grow. Such cross-protective immunity against unrelated gliomas suggests that mM-CSF transfected tumor cells have immunotherapeutic potential for use as an allogeneic tumor vaccine.

Rat glioma cell death induced by cationic liposome-mediated transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir treatment.

We studied antitumor effects and cell death induced by cationic liposome-mediated gene transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir treatment in cultured rat T9 glioma cells and in experimental gliomas produced from this cell line. To transfer genes we used small unilamellar cationic liposomes containing N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride. Video-enhanced contrast differential interference contrast microscopy was used for morphologic observations of cultured cells. When we treated the cells or implanted gliomas with the liposomes and ganciclovir, a strong effect was seen against tumor cells, and survival of tumor-implanted rats was increased. Morphologically, cell death observed after HSV-tk gene/liposome and ganciclovir treatment in the cultured glioma cells included both apoptosis and necrosis. Introduction of the HSV-tk gene in a DNA-liposome complex followed by ganciclovir treatment induced both apoptosis and necrosis, which together resulted in a potent antitumor effect.

Macrophages that kill glioma cells expressing the membrane form of macrophage colony stimulating factor are resistant to prostaglandin E2 and interleukin-10

Malignant rat T9 glioma cells retrovirally transduced with the membrane form of macrophage colony stimulating factor (mM-CSF) were killed by bone marrow derived macrophages in 24 h cytotoxicity assays. Prostaglandin E2 (PGE) and interleukin-10 (IL10) were tested for their ability to block this tumoricidal reaction. Only at very high nonphysiological concentrations of PGE (10 −5 and 10 −6 M) was this cytotoxicity inhibited. Use of high doses of theophylline, a phosphodiesterase inhibitor, also prevented macrophages from killing the mM-CSF transduced target cells. IL10 did not alter the killing potential of the mM-CSF tumoricidal macrophages, even though IL10 reduced the production of nitric oxide by macrophages in response to tumor necrosis factor and lipopolysaccharide. IL10 enhanced the growth of bone marrow macrophages suggesting that IL10 has a complex role in the regulation of tumoricidal macrophages. Thus, the mM-CSF may be an ideal agent to treat tumors that utilize either of these two immunosuppressive defense mechanisms that may block other forms of treatment.

Macrophages Kill T9 Glioma Tumor Cells Bearing the Membrane Isoform of Macrophage Colony Stimulating Factor Through a Phagocytosis-Dependent Pathway

Rat T9 glioma cells transfected with the gene for the membrane isoform of macrophage-CSF (mM-CSF) but not for the secreted isoform of M-CSF were directly killed by bone marrow-derived macrophages. Macrophage-mediated cytolysis of the mM-CSF-transfected clone was blocked by using chemical inhibitors of phagocytosis such as iodoacetate, 2-deoxyglucose, gadolinium chloride, and cytochalasin B. In contrast, macrophage-mediated killing of mM-CSF-expressing tumor cells was augmented by the microtubule inhibitor, colchicine. Use of nitric oxide and reactive oxygen intermediate inhibitors failed to alter the macrophage-mediated killing of the mM-CSF-transfected tumor cells. Photomicroscopy, using immunohistochemical staining with the anti-Hck Ab to distinguish macrophages from tumor cells, revealed that phagocytosis began within 2 h after addition of the mM-CSF-bearing tumor cells. Photocinematography confirmed that macrophages first phagocytosized and then lysed the internalized mM-CSF transfectant cells. Using annexin V and acridine orange staining techniques, macrophages phagocytosized living mM-CSF-transfected tumor cells.

Macrophages Can Recognize and Kill Tumor Cells Bearing the Membrane Isoform of Macrophage Colony-Stimulating Factor

NBXFO hybridoma cells produced both the membrane and secreted isoforms of macrophage colony-stimulating factor (M-CSF). Murine bone marrow cells stimulated by the secreted form of M-CSF (sM-CSF) became Mac1+, Mac2+, Mac3+, and F4/80+ macrophages that inhibited the growth of NBXFO cells, but not L1210 or P815 tumor cells. In cytotoxicity studies, M-CSF activated macrophages and freshly isolated macrophages killed NBXFO cells in the presence of polymyxin B, eliminating the possibility that contaminating lipopolysaccharide (LPS) was responsible for the delivery of the cytotoxic signal. Retroviral-mediated transfection of T9 glioma cells with the gene for the membrane isoform of M-CSF (mM-CSF), but not for the secreted isoform of M-CSF, transferred the ability of macrophages to kill these transfected T9 cells in a mM-CSF dose-dependent manner. Macrophage-mediated killing of the mM-CSF transfected clone was blocked by using a 100-fold excess of recombinant M-CSF. Catalase, superoxide dismutase, and the nitric oxide inhibitor, N-omega-nitro-arginine methyl ester (NAME), did not effect macrophage cytotoxicity against the mM-CSF transfectant T9 clones. T9 parental cells when cultured in the presence of an equal number of the mM-CSF transfectant cells were not killed, indicating specific target cell cytotoxicity by the macrophages. Electron microscopy showed that macrophages were capable of phagocytosizing mM-CSF bearing T9 tumor cells and NBXFO hybridoma cells; this suggested a possible mechanism of this cytotoxicity. This study indicates that mM-CSF provides the necessary binding and triggering molecules through which macrophages can initiate direct tumor cell cytotoxicity.

Activation of the Sphingomyelin Cycle Through the Low-Affinity Neurotrophin Receptor

The role of the low-affinity neurotrophin receptor (p75NTR) in signal transduction is undefined. Nerve growth factor can activate the sphingomyelin cycle, generating the putative-lipid second messenger ceramide. In T9 glioma cells, addition of a cell-permeable ceramide analog mimicked the effects of nerve growth factor on cell growth inhibition and process formation. This signaling pathway appears to be mediated by p75NTR in T9 cells and NIH 3T3 cells overexpressing p75NTR. Expression of an epidermal growth factor receptor-p75NTR chimera in T9 cells imparted to epidermal growth factor the ability to activate the sphingomyelin cycle. These data demonstrate that p75NTR is capable of signaling independently of the trk neurotrophin receptor (p140trk) and that ceramide may be a mediator in neurotrophin biology.

Ceramide activates heterotrimeric protein phosphatase 2A

Ceramide activates a cytosolic protein phosphatase present in rat T9 glioma cells and rat brain. Ceramide-activated protein phosphatase (CAPP) was found to share several properties with protein phosphatase 2A (PP2A) leading to the hypothesis that ceramide may directly activate PP2A. PP2A was isolated as a heterotrimer (AB'C, AB alpha C), heterodimer (AC), or free C subunit, and the effect of ceramide on the catalytic activity was assessed. C2-ceramide, 5-20 microM, activated heterotrimeric PP2A up to 3.5-fold but had no effect on the activity of AC or C. Ceramides possessing hexanoyl, decanoyl, and myristoyl but not stearoyl acyl chains also activated heterotrimeric PP2A. Ceramide activation of heterotrimeric PP2A required the presence of a B subunit since trypsinization or heparin treatment abolished ceramide activation. Activation of heterotrimeric PP2A was specific for ceramide because related sphingolipids had no effect. Moreover, dihydro-C2-ceramide, which lacks the trans double bond in the sphingoid base, inhibited AB'C activity by > 90% at 10 microM. The specificity of activation of AB'C and AB alpha C by stereoisomers of C2-ceramide was found to differ. Whereas activation of AB'C by either DL-erythro- or threo-C2-ceramide was similar, AB alpha C was activated by either D- or L-erythro-C2-ceramide but not by the threo isomers. CAPP isolated from T9 cells was most effectively activated by D-erythro-C2-ceramide. CAPP was found to possess two peaks of ceramide activated phosphatase activity. The initial peak of activity was coincident with the elution of AB'C and was stimulated 1.8-fold by 20 microM C2-ceramide. A second peak of phosphatase activity was negligible in the absence of ceramide but was stimulated 5.5-fold by 20 microM C2-ceramide. These results support the hypothesis that ceramide is a specific lipid second messenger modulating heterotrimeric PP2A activity.

Photodynamic Therapy Using Pheophorbide a and Nd:YAG Laser

The authors describe a new photodynamic therapy (PDT) method for malignant brain tumors. Pheophorbide a (Ph-a), the photosensitizer, has low toxicity, causes no skin sensitization and is activated with an acoustic Q switched neodymium yttrium-argon-garnet (Nd:YAG) laser which achieves deep tissue penetration. The Ph-a distribution in Fisher 344 (F344) rats bearing rat T9 glioma at 24 hours after intravenous injection was very low in the normal brain tissue, but significantly higher in the T9 glioma giving a tumor to normal brain tissue concentration ratio of 7.5:1. The in vitro survival rate of T9 glioma cells pretreated with Ph-a was 68.8 +/- 5.4% after laser irradiation for 20 minutes, significantly lower than in the control groups. This indicates that Ph-a was activated with the acoustic Q switched Nd:YAG laser causing the photodynamic effect. The survival rate after Ph-a pretreatment and laser irradiation in a waterbath at 44.0 degrees C was further reduced to 15.8 +/- 3.3%. In vivo PDT studies using T9 glioma cells inoculated into the dorsal region of F344 rats showed tumor eradication in four of six rats. The combination of PDT and laser hyperthermia produced tumor eradication in all six rats. The combination of PDT and hyperthermia is a promising method for tumor treatment.

Induction of glutathione S-transferase, placental type in T9 glioma cells by dibutyryladenosine 3?,5?-cyclic monophosphate and modification of its expression by naturally occurring isothiocyanates

The effect of dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl cAMP) on the expression of glutathione S-transferase placental type (GST-P) was examined in rat glioma cell line using an immunohistochemical technique. Cultured T9 glioma cells were negative for GST-P activity under normal conditions. However, treatment with 1 mM dibutyryl cAMP produced GST-P expression in about 50% of the cells, as well as some morphological changes. The expression of GST-P was increased with addition of dibutyryl cAMP together with 1 microgram/ml allyl isothiocyanate (AITC) or 0.1 microgram/ml benzyl isothiocyanate (BITC). With these combinated treatments, almost all cultured cells showed a strong positive reaction for GST-P, although AITC or BITC alone elicited GST-P in only 5% of the cultured cells. The results of the present study indicate that dibutyryl cAMP causes functional as well as morphological differentiation of T9 glioma cells.