D54-MG Cells Research Articles

Centrosome amplification induced by survivin suppression enhances both chromosome instability and radiosensitivity in glioma cells

Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.

Participation of the Chaperone Hsc70 in the Trafficking and Functional Expression of ASIC2 in Glioma Cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.

Xanthine Oxidase–Dependent Regulation of Hypoxia-Inducible Factor in Cancer Cells

During chemical hypoxia induced by cobalt chloride (CoCl2), hypoxia-inducible factor 1alpha (HIF1-alpha) mediates the induction of a variety of genes including erythropoietin and vascular endothelial growth factor. We used glioma cells with oxidative phosphorylation-dependent (D54-MG) and glycolytic-dependent (U251-MG) phenotypes to monitor HIF1-alpha regulation in association with redox responsiveness to CoCl2 treatment. We showed that CoCl2 increased xanthine oxidase (XO)-derived reactive oxygen species (ROS), which causes accumulation of HIF1-alpha protein in U251-MG cells. Under these conditions, blockade of XO activity by pharmacologic (N-acetyl-L-cysteine or allopurinol) or molecular (by small interfering RNA) approaches significantly attenuated HIF1-alpha expression. Exogenous H2O2 stabilizes HIF1-alpha protein. XO was present in these cells and was the primary source of free radicals. We also showed higher XO activity in cells exposed to CoCl2 compared with cells grown in normoxia. From the experiments shown here, we concluded that ROS were indeed generated in D54-MG cells exposed to CoCl2 but it was unlikely that ROS participated in the hypoxic signal transduction pathways in this cell type. Possibly, cell type-dependent and stimulus-dependent factors may control ROS dependency or redox sensitivity of HIF1-alpha and thus HIF1-alpha activation either directly or by induction of specific signaling cascades. Our findings reveal that XO-derived ROS is a novel and critical component of HIF1-alpha regulation in U251-MG cells, pointing toward a more general role of this transcription factor in tumor progression.

947. Gene Directed Enzyme/Prodrug Therapy of Human Glioma Xenografts Using Mutant Escherichia coli Cytosine Deaminase

Combined treatment using suicide gene therapy and radiation therapy has the potential to become a powerful new method of cancer therapy. We have developed a non-replicative adenoviral vector encoding a mutant bacterial cytosine deaminase (bCD) gene harboring substitution of an alanine (A) for the aspartic acid (D) at position 314 in the CD protein (AdCD-D314A) which has a higher affinity for 5-fluorocytosine (5-FC) than wild type bCD (CDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdCD-D314A with the prodrug 5-FC and radiation treatment (RT) against human glioma. The results of CD enzyme activity assays showed that the conversion of 3H-5-FC to 3H-5-FU was elevated 183.3, 205.6 and 102.2-fold in D54MG, U87MG and U251MG human glioma cells, respectively, for cells infected with 2 multiplicity of infection (MOI) of AdCD-D314A compared to AdCDwt. AdCD-D314A infection also resulted in increased 5-FC-mediated cell killing. The 50% inhibitory concentration produced by 5-FC decreased by 7.8-fold for D54MG, 32.9-fold for U87MG, and 8.1-fold for U251MG cells infected with 10 MOI of AdCD-D341A in comparison with AdCDwt. Animal studies showed significant inhibition of tumor growth of D54MG glioma subcutaneous xenografts by the combination of AdCD-D314A/5-FC with RT as compared with either agent alone, or with AdCDwt/5-FC plus RT. The results demonstrate that the combination of AdCD-D314A/5-FC with radiation produces markedly increased cytotoxicity in cancer cells in vitro and in vivo compared to AdCDwt. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for glioma therapy.

Silatecan DB-67 is a novel DNA topoisomerase I–targeted radiation sensitizer

The silatecan 7-tert-butyldimethylsilyl-10-hydroxy-camptothecin (DB-67) represents a new generation of camptothecin derivatives that exhibits a potent in vitro DNA topoisomerase I (TOP1)-mediated DNA-damaging activity, improved blood stability, and holds significant promise for the treatment of human cancers. In this study, we characterize the role of TOP1 in mediating the radiosensitization activity of DB-67. As examined by clonogenic survival assay, DB-67 exhibited potent radiosensitization activity at a concentration 10-fold lower than camptothecin in the human glioma D54-MG and T-98G cells, which harbor wild-type and mutant p53, respectively. Analyzed by the single-hit multitarget model, DB-67 induced radiosensitization by obliterating the "shoulder" of the radiation survival curve in the D54-MG cells. The in vivo targeting of TOP1 by DB-67 was investigated by immunoblot analysis. In a dose-dependent manner, DB-67 specifically stimulates covalent linking of TOP1 to chromosomal DNA at concentrations 10-fold lower than camptothecin in the D54-MG cells. The potency of in vivo targeting of TOP1 by DB-67 correlates well with its cytotoxicity and radiosensitization activity. Furthermore, DB-67 exhibited substantially less cytotoxicity and radiosensitization activity in the TOP1 mutant Chinese hamster lung fibroblast DC3F/C-10 cells than in their parental DC3F cells. Together, our data show that DB-67 exhibits potent cytotoxicity and radiosensitization activity by targeting TOP1 in mammalian cells and has great potential for being developed to treat human cancers.

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.

607. In Vitro and In Vivo Monitoring of 5-Fluorosytosine Metabolism with 19F-NMR Spectroscopy after Adenoviral Transduction of Cells with Bifunctional Yeast CDUPRT Fusion Gene

Transfer into tumor of genes is an attractive gene therapy strategy for selective elimination of cancer cells. One of the newly invented suicide gene encodes bifunctional yeast CDUPRT fusion protein, which combines the enzymatic activity of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT). This protein efficiently catalyzes the direct conversion of 5-FC (a non-toxic prodrug) into the toxic metabolites 5-fluorouracil (5-FU) and its toxic derivative 5-fluorouridine-5'-monophosphate (FUMP). This study was undertaken to evaluate the applicability of 19F-MRS for monitoring in vitro and in vivo of the conversion of 5-fluorocytosine (5-FC) to fluorouracil after adenovirus mediated transfer of a CDUPRT Fusion Gene. For this purpose a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the yeast CDUPRT gene in E1 region was constructed. We employed both 19F-MRS and cell biology techniques to measure the pharmacokinetics and cytotoxicity of 5-FC for infected human glioma D54MG cells.

Plasminogen activator inhibitor-1 increases the expression of VEGF in human glioma cells

The level of plasminogen activator inhibitor-1 (PAI-1) in tumor tissue has been shown to be an independent negative prognostic factor in different cancers. There are several proposed reasons for this, among these, the influence of PAI-1 on tumor neovascularization and cell migration. We report that PAI-1 stimulates expression and release of vascular endothelial growth factor (VEGF) in the human glioma cell line D54Mg, and thereby stimulates the proliferation of human umbilical vein endothelial cells (HUVEC) in vitro. To search for possible molecular effects of PAI-1 on malignant cells, cDNA array hybridization analysis of D54Mg glioma cells transfected with an adenoviral PAI-1 expression vector was performed. This revealed that the VEGF response was accompanied with the simultaneous upregulation of GADD153, Rho GTPase activating protein 4 (p115), Collagen type VI alpha 1 and cell division cycle 42 (CDC42) transcripts. Exogenous treatment of D54Mg cells with a constitutively active recombinant PAI-1 protein confirmed an upregulation of VEGF expression in a time- and dose-dependent manner, and supernatants from such cultures stimulated the proliferation of human umbilical vein endothelial cells in vitro. In 44 human glioma biopsies, patients, the protein levels of PAI-1 correlated strongly with the levels of VEGF in the tumor tissues. Whereas VEGF expression correlated inversely with survival, there was no statistically significant prediction of survival by PAI-1 in this group of patients. These clinical data support and strengthen the hypothesis that PAI-1 is one of the factors regulating and inducing the VEGF expression in human gliomas. The induction of VEGF expression and thus endothelial cell proliferation may represent an as yet undiscovered mechanism whereby PAI-1 contributes to tumor neoangiogenesis.

Photodynamic inhibition of enzymatic detachment of human cancer cells from a substratum.

Photodynamic therapy (PDT) is currently used for cancer treatment. It is shown that sublethal PDT of human WiDr adenocarcinoma cells and D54Mg glioblastoma cells with 5-aminolevulinic acid (ALA), disulfonated tetraphenylporphyrine (TPPS(2a)), or MitoTracker Red (MTR) inhibits their trypsin-induced detachment from a plastic substratum. TPPS(2a) was bound selectively to the plasma membrane, whereas MTR was found in mitochondria. Both granular and diffuse fluorescence of ALA-derived protoporphyrin IX (PpIX) was observed in the perinuclear cytoplasm but not in the plasma membrane of WiDr cells stained for 2 h with 1 mM ALA. In D54Mg cells, PpIX fluorescence was observed not only in the cytoplasm but also in the plasma membrane. Fluorescence measurements showed a progressive accumulation of PpIX in the WiDr cells during incubation with ALA and a PpIX efflux into the medium after 1 h or longer incubation. PpIX retained in the plasma membrane during efflux may be responsible for PDT-induced impairment of cell adhesion. On the other hand, MTR-PDT or ALA-PDT after 15-min incubation, when the newly synthesized PpIX should remain in mitochondria, also inhibited enzymatic cell detachment. Therefore, photodynamic targeting of mitochondria, remote from the cell surface where adhesion occurs, may disturb cell adhesion. Photodynamic inhibition of enzymatic cell detachment may be related to PDT-induced inhibition of tumour metastasis.

Modulation of glioma cell invasion and motility by adenoviral gene transfer of PAI-1.

A number of studies have emphasized the role of PAI-1 as an important regulator of tumor cell invasion and metastasis. The hallmark of primary tumors of the central nervous system and glioblastomas in particular is the diffuse invasion into the normal brain tissue. Since PAI-1 is expressed in such tumors, we studied the effect of adenoviral-mediated transfer of the PAI-1 gene in regulating the in vitro invasiveness of D54Mg glioma cells into Matrigel, and into fetal rat brain aggregates. Treatment of D54Mg cells with 50 MOI (multiplicity of infection) of the replication defective vector AdCMVPAI-1 increased PAI-1 expression 23-fold compared to control vectors, and the invasion through Matrigel was reduced by 67%. The motility of the cells was reduced by 58% compared to controls (indicating that inhibition of motility was the principal effect of PAI-1 in these cells). The ability of D54Mg tumor spheroids to invade fetal rat brain aggregates was not reduced by the PAI-1 gene transfer. The results show that overexpression of PAI-1 can inhibit glioma cell motility and invasion through extracellular matrix (ECM) components, like laminin and collagen, but does not inhibit tumor cell invasion in a three-dimensional invasion assay, simulating normal brain tissue having a different ECM and interstitial composition. The different results obtained in the two invasion assays reflect the complex biological effects of the uPA/PAI-1 system, and questions a simplistic view of PAI- I as an inhibitor of brain tumor invasion.

Potential of adenoviral p53 gene therapy and irradiation for the treatment of malignant gliomas.

We investigated the combined effects of p53 gene transfer and irradiation and its still unclear interaction mechanism in human gliomas. Four human glioma cell lines expressing mutant type p53 (U373 and A172) and wild-type p53 (D54MG and EFC-2) were transfected by adenoviral vectors bearing p53 gene at 50 multiplicity of infection. Two days after transfection, cells were irradiated (3, 6, and 9 Gy). The cytotoxicity was evaluated by clonogenic assay. The quantitative analysis of apoptosis and cell cycle analysis were performed using flow cytometry. Irradiation combined with adenoviral p53 transfection significantly increased cytotoxicity, which was additive in cell lines with wild-type p53 and more than additive in cell lines with mutant p53. The combination of two modalities increased the apoptotic population by 14% in A172 cells and 20% in D54 MG cells, which were the sum of apoptosis from each modality. Adenoviral p53 transfection increased the G1 phase fraction and concomitant decrease of radioresistant S phase fraction in A172 and D54MG cells. Our study demonstrated that p53 gene transfer combined with irradiation increased absolute cytotoxicity in human glioma cells used in this experiment. The interaction mechanism for increased cytotoxicity involved, in part, increased apoptosis and change of cell cycle profile.

Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status

During aerobic metabolism, a small amount of partially reduced oxygen is produced, yielding reactive oxygen species (ROS). Peroxisomes and mitochondria are major contributors to cellular ROS production, which is normally balanced by consumption by antioxidants. The fatty acid analogue tetradecylthioacetic acid (TTA) promotes mitochondrial and peroxisomal proliferation, and may induce oxidative stress and change the growth potential of cancer cells. In the present study, we found that TTA reduced [ 3H]thymidine incorporation in the glioma cell lines BT4Cn (rat), D54Mg (human), and GaMg (human) in a dose- and time-dependent manner. The 50% inhibitory TTA doses were approximately 125 μM for BT4Cn and D54Mg cells and 40 μM for GaMg cells after 4 days. α-Tochopherol counteracted this inhibition in GaMg cells. TTA enhanced the oxidation of [1- 14C]palmitic acid, which could be explained by stimulation of enzymes involved in peroxisomal (fatty acyl-CoA oxidase) and/or mitochondrial (carnitine palmitoyltransferase) fatty acid oxidation. The glutathione content and the activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase were differentially affected. Increased malondialdehyde (MDA) production was seen in TTA-treated GaMg and D54Mg cells, but not in BT4Cn cells, in vitro. In BT4Cn tumor tissue from TTA-treated rats, MDA was increased while the α-tocopherol content tended to decrease. TTA increased the level of cytosolic cytochrome c in BT4Cn cells, which suggests induction of apoptotic cascades. Although several mechanisms are likely to be involved in the TTA-mediated effects on growth, we propose that modulation of cellular redox conditions caused by changes in fatty acid metabolism may be of vital importance.

Transforming growth factor-beta 1 inhibits inflammatory cytokine-induced C3 gene expression in astrocytes.

In this report, we show that transforming growth factor-beta (TGF-beta) can significantly inhibit the capacity of IFN-gamma, IL-1 beta, and TNF-alpha to augment expression of the central component of complement C3 in the human astroglioma cell line D54-MG. Treatment of D54-MG cells with TGF-beta alone had no dose- or time-dependent effect on basal C3 protein or mRNA levels. However, TGF-beta suppressed induction of C3 expression at both the protein and mRNA level in D54-MG cells treated with inflammatory cytokines. The extent of TGF-beta-mediated suppression was cytokine-specific, and suppression of protein production did not necessarily correspond to reductions in steady-state mRNA levels for each cytokine. Similar findings were obtained at the mRNA level using primary rat astrocytes, indicating that TGF-beta can modulate C3 gene expression in nontransformed astrocytic cells. Kinetic studies demonstrated that TGF-beta mediates its suppressive effect for at least 72 h, and that pretreatment of cells with TGF-beta for as little as 2 h significantly reduced the ability of all three inflammatory cytokines to enhance C3 expression. Our results suggest that TGF-beta may play an important role in modulating the endogenous synthesis of complement by astrocytes under inflammatory conditions.

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells , demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells.

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1 beta or TNF-alpha, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1 beta or TNF-alpha, respectively. D54-MG cells require a minimal 1 h exposure to IL-1 beta in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-alpha. Simultaneous treatment of D54-MG cells with IL-1 beta and interferon-gamma (IFN-gamma) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-alpha and IFN-gamma. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1 beta (5.3-fold increase) and TNF-alpha (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1 beta and IFN-gamma. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-gamma, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.

Interferon-gamma regulation of C3 gene expression in human astroglioma cells

In this report, we show that the human astroglioma cell line, D54-MG, constitutively expresses C3 mRNA and secretes antigenically detectable C3 protein. the cytokine interferon-gamma (IFN-γ) enhances C3 mRNA and protein expression by D54-MG cells in a dose- and time-dependent manner. C3 mRNA from both D54-MG cells and primary human adult astrocytes has the same apparent size (5.1–5.2 kb) as C3 mRNA from hepatocyte and monocyte cell lines. Constitutive C3 mRNA levels in D54-MG cells and primary human astrocytes are comparable. Primary rat astrocytes also constitutively express C3 mRNA, which is enhanced upon exposure to IFN-γ. These data are novel since expression of C3 in other cell types is refractory to IFN-γ. In the central nervous system (CNS), endogenous complement production by astrocytes, and enhancement by the cytokine IFN-γ, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis (MS).

Tumor necrosis factor production and receptor expression by a human malignant glioma cell line, D54-MG

Human malignant gliomas possess some of the same immune-related functions as astrocytes do. For instance, they are capable of secreting various immunoregulatory molecules and expressing HLA-DR antigens on their surface. The human malignant glioma cell line, D54-MG, was used to investigate the proliferative effects of tumor necrosis factor-alpha (TNF-alpha) and the expression of specific surface receptors for TNF-alpha. Additionally, we were interested in examining whether D54-MG cells are capable of synthesizing and secreting biologically active TNF-alpha. D54-MG cells responded in a mitogenic fashion upon incubation with TNF-alpha for 48 h under serum-free conditions. 125I-labeled TNF-alpha was used in this study to investigate the expression of receptors specific for TNF-alpha on D54-MG cells. Scatchard analysis of our receptor binding data produced curvilinear plots indicating there are two distinct receptor sites for TNF-alpha. From these data, we calculated that there are approximately 3500 high affinity and 24,666 low affinity binding sites per cell. Pretreating these cells with interferon-gamma (IFN-gamma) resulted in a 2-fold increase in the number of high affinity binding sites and a moderate increase in the number of low affinity binding sites, with no appreciable change in binding affinity (Kd) of either site. D54-MG cells were unable to constitutively secrete TNF-alpha; however, upon stimulation, these cells synthesize and secrete biologically active TNF-alpha. Polyclonal antisera reactive with human macrophage-derived TNF-alpha neutralized the cytotoxicity of D54-MG-derived TNF-alpha, demonstrating that the cytotoxic activity was in fact due to TNF-alpha. Our observations indicate that TNF-alpha could act in an autocrine fashion to induce the proliferation of this malignant glioma cell line and that TNF-alpha exerts its effect by binding to specific TNF-alpha receptors whose expression was enhanced by IFN-gamma.