C6 Rat Glioma Cell Line Research Articles

Gliomas in rodent whisker barrel cortex: a new tumor model

Object Surgical treatment of gliomas is difficult because they are invasive. Invasion of essential cortex often limits or precludes surgical resection. A tumor model was developed in which the rodent whisker barrel cortex was used to examine how gliomas affect cortical function and structure. Methods Both DBT (mouse) and C6 (rat) glioma cell lines were grown in culture and labeled with the fluorescent marker Dil in vitro. Labeled tumor cells were then injected into the whisker barrel cortex of adult mice and rats. Neurological assessments were made daily and magnetic resonance (MR) images were obtained. Animals were killed by perfusion 6 to 14 days after injection, and histological sections were prepared and studied. Tumors were found in all 20 rats and 10 mice that had been injected with the C6 and DBT cell lines, respectively. The animal cells had been labeled with Dil in vitro, and all in vivo tumors proved to be Dil positive. The MR images revealed the tumor locations and serial MR images demonstrated tumor growth. Histological evaluation confirmed the location of the tumor and the disruption of barrel cortex architecture. Conclusions Both DBT and C6 glioma cell lines can be used to generate malignant glial tumors reproducibly in the whisker barrel cortex. Fluorescent labeling and cytochrome oxidase staining permit visualization of tumor growth patterns, which disrupt the barrel cortex by microscopic invasion and by gross tissue deformation. Magnetic resonance imaging demonstrates the anatomical extension of these tumors in live rodents. Using this model for further studies on the effects of malignant glioma growth on functional cerebral cortex should advance our understanding of the neurological issues and management of patients with these tumors.

Expression levels of the DNA repair enzyme HAP1 do not correlate with the radiosensitivities of human or HAP1-transfected rat cell lines.

Apurinic/apyrimidinic (AP) sites in DNA are potentially lethal and mutagenic. They can arise spontaneously or following DNA damage from reactive oxygen species or alkylating agents, and they constitute a significant product of DNA damage following cellular exposure to ionizing radiation. The major AP endonuclease responsible for initiating the repair of these and other DNA lesions in human cells is HAP1, which also possesses a redox function. We have determined the cellular levels of this enzyme in 11 human tumour and fibroblast cell lines in relation to clonogenic survival following ionizing radiation. Cellular HAP1 levels and surviving fraction at 2 Gy (SF2) varied five- and tenfold respectively. However, no correlation was found between these two parameters following exposure to γ-irradiation at low (1.1 cGy per min) or high (108 cGy per min) dose rates. To examine this further, wild-type and mutant versions of HAP1 were overexpressed, using an inducible HAP1 cDNA expression vector system, in the rat C6 glioma cell line which has low endogenous AP endonuclease activity. Induction of wild-type HAP1 expression caused a > fivefold increase in the capacity of cellular extracts to cleave an oligonucleotide substrate containing a single abasic site, but increased expression did not confer increased resistance to γ-irradiation at high- or low-dose rates, or to the methylating agent methyl methanesulphonate (MMS). Expression in C6 cell lines of mutant forms of HAP1 deleted for either the redox activator or DNA repair functions displayed no apparent titrational or dominant negative effects. These studies suggest that the levels of endogenous AP endonuclease activities in the various cell lines examined are not limiting for efficient repair in cells following exposure to ionizing radiation or MMS. This contrasts with the correlation we have found between HAP1 levels and radiosensitivity in cervix carcinomas (Herring et al (1998) Br J Cancer 78: 1128–1133), indicating that HAP1 levels in this case assume a critical survival role and hence that established cell lines might not be a suitable model for such studies. © 1999 Cancer Research Campaign

Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-γ caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-γ-induced expression of iNOS, but not interferon regulatory factor-1 (IRF-1) mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-γ and any further induction was blocked by protein synthesis inhibitor cycloheximide (CHX). FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action.

Modulation of TNF-α mRNA production in rat C6 glioma cells by TNF-α, IL-1β, IL-6, and IFN-α: In vitro analysis of cytokine-cytokine interactions

Cytokines regulate the expression of other cytokines in the centrally derived rat C6 glioma cell line. However, the modulation of tumor necrosis factor-α (TNF-α, a pivotal proinflammatory cytokine) in C6 cells is unknown. Here we investigated the expression of TNF-α mRNA in C6 glioma cells in response to TNF-α, interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), and interferon-α (IFN-α). The data show that (1) IL-1β induced a significant upregulation of TNF-α mRNA; (2) the effect of IL-1β on TNF-α mRNA expression was completely blocked by the concomitant application of IL-1Ra, which suggests specificity of IL-1β action through the IL-1 signaling receptor; (3) no detectable modulation of TNF-α mRNA expression was observed with the individual applications of TNF-α, IL-6, or IFN-α; (4) the concomitant treatments of TNF-α + IL-1β or TNF-α + IL-1β + IL-6 strongly upregulated TNF-α mRNA expression, whereas the concomitant application of TNF-α + IL-6 or IL-1β + IL-6 induced a moderate increase; and (5) IFN-α significantly attenuated induction of TNF-α mRNA by TNF-α + IL-1β + IL-6. Thus, IL-1β, TNF-α and IL-6 interact to upregulate TNF-α mRNA expression synergistically, and IFN-α acts as an inhibitory cytokine in C6 glioma cells. These findings also suggest that the rat C6 glioma cell line may be used as an in vitro model to characterize cytokine-cytokine interactions.

Inhibition of p42/p44 mitogen-activated protein kinase by oxysterols in rat astrocyte primary cultures and C6 glioma cell lines.

We previously demonstrated that oxysterols inhibit the growth of experimental glioblastoma induced in the rat brain cortex. Mechanism of action of these compounds remains obscure. In this study, we investigated the effect of 7beta-hydroxycholesterol (7beta-OHCH) and 7ketocholesterol (7k-CH) on the growth and MAP kinase activity in three in vitro biological models: rat astrocyte primary cultures, primary cultures treated by dibutyryl-cAMP (reactive cells), and the C6 glioma cell line. The oxysterols are not lethal to primary astrocytes, even if MAP kinase activity is decreased, particularly when cells were treated with 7k-CH. Both oxysterols are toxic to reactive astrocytes, and as compared with untreated primary cultures, they amplified the MAP kinase activity decrease. However, the mechanism of action of oxysterols on reactive astrocytes seems not to be linked to the MAP kinase pathway. In highly proliferating C6 cell lines, only 7beta-OHCH has an antiproliferative effect and is cytotoxic. The inhibition of MAP kinase activity is a function of 7beta-OHCH concentration. PD098059, a MAP kinase pathway inhibitor, has only a time-limited antiproliferative effect on C6 cell growth. We conclude that in C6 cells, the MAP kinase activity decrease is correlated with the toxic effect of 7beta-OHCH and occurs at first stages of 7beta-OHCH action.

Inhibition of p42/p44 mitogen‐activated protein kinase by oxysterols in rat astrocyte primary cultures and C6 glioma cell lines

Inhibition of p42/p44 mitogen‐activated protein kinase by oxysterols in rat astrocyte primary cultures and C6 glioma cell lines

Thymosin fraction 5 inhibits the proliferation of the rat neuroendocrine MMQ pituitary adenoma and C6 glioma cell lines in vitro.

Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5-50 x 10(3) cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100-500 microg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 microg/ml TF5, whereas the highest concentration of this preparation (i.e. 500 microg/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 x 10(3) MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 10(3) cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1-1000 nM) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10-30 x 10(3) cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 microg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 microg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin alpha1 (T alpha1), thymosin beta4 (T beta4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.

Thymosin Fraction 5 Inhibits the Proliferation of the Rat Neuroendocrine MMQ Pituitary Adenoma and C6 Glioma Cell Lines in Vitro

Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5–50 × 103 cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100–500 μg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 μg/ml TF5, whereas the highest concentration of this preparation (i.e. 500μ g/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 × 103 MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 × 103 cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1–1000 nm) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10–30 × 103 cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 μg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 μg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin α1 (Tα1), thymosinβ 4 (Tβ4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH-cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. In previous studies, we have used the rat glioma C6 cell line as an in vitro model system and identified the presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 by reverse transcription followed by polymerase chain reaction (RT–PCR). In C6 cells, the induction of P450 1A1 and 2B1/2 at mRNA level after BA (benzo(a)anthracene) or PB (phenobarbital) treatments was detected. In this study, analysis of microsomal preparations of glioma C6 cells was utilized to demonstrate the presence of P450 2B and P450 reductase at the protein level. ELISAs showed that PB induced P450 2B proteins 12-fold. These experiments further establish that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system that can be induced by P450 inducers. We also found that the mRNAs of P450 1A1 and 2B1/2 from glioma C6 cells do not bind to the oligo(dT)-based separation techniques efficiently, suggesting that they may have very short poly(A) tails. The half-lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are 1/10 and 1/3 of that in liver, respectively. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half-lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors.

Differentiation-dependent expression of transgenes in engineered astrocyte cell lines

The utility of transgenes for both basic and applied studies has been augmented by the recent advent of versions that can be regulated by the addition of suitable activators. However, still more convenient would be transgenes whose expression responded appropriately to endogenous signals. The promoter of the glial fibrillary acidic protein (GFAP) gene is a candidate for this role in the central nervous system (CNS) since the GFAP gene is specifically expressed in astrocytes in the CNS and its activity is upregulated in response to almost any CNS injury. As a feasibility study, we isolated a C6 rat glioma cell line stably transfected with a lacZ reporter gene driven by the gfa2 human GFAP promoter fragment. We find that the activity of the transgene indeed responds to an environmental signal, forskolin, that induces astrocyte-like differentiation of C6 cells. We also isolated a C6 line carrying a transgene in which the gfa2 promoter directs expression of a cDNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis. This transgene should be of considerable interest for gene therapy for Parkinson's disease. We show that in this cell line both TH mRNA and protein are upregulated by forskolin. Finally, we note that the growth rate of C6 cells is severely depressed by forskolin, suggesting that predifferentiation of these cells prior to implant may retard their tumor forming capacity, prolonging the time that they can be used in animal models in vivo.

Dipeptidyl peptidase IV in C6 rat glioma cell line differentiation.

The C6 rat glioma cell line is broadly used as a model in studies of glial cell differentiation. In the present study we demonstrated a significantly higher total cellular, but especially membrane-associated, activity of dipeptidyl peptidase IV in differentiated C6 cells in comparison with their proliferating counterparts. The majority, but not all, of enzyme isoelectric focusing isoforms from differentiated C6 cells displayed a substantially higher activity compared to the proliferating cells, with G-P-NHMec as the substrate. Non-denaturing polyacrylamide gradient gel electrophoresis showed the presence of one major peak of activity, dipeptidyl peptidase IV (Mr of about 220000), in both proliferating and differentiated C6 cells. The results indicate that dipeptidyl peptidase IV regulation is associated with C6 rat glioma cell differentiation.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside

Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promoters of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Expression and induction of cytochrome P-450 1A1 and P-450 2D subfamily in the rat glioma C6 cell line

The cytochrome P-450 ( P-450) monooxygenase system can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs and pollutants. The functions of this system are as diverse as the substrates. Though this enzyme system has the highest level of activity in the liver, it is present in other tissues, including brain. In this study, we have established the rat glioma C6 cell line as an in vitro model system to examine the expression and induction of P-450 1A1 and the P-450 2D subfamily. Rat glioma C6 cells were treated with P-450 inducers phenobarbital (PB) or benzo[ a]anthracene (BA). The presence of P-450 1A1 and 2D1–5 was detected by reverse transcription followed by polymerase chain reaction (RT–PCR) and confirmed by restriction enzyme digestion. The induction of P-450 1A1 and 2D1–5 was quantified using competitive PCR. Although P-450 2D1–5 do not seem to be affected by PB or BA treatment, tenfold induction of P-450 1A1 mRNA after BA treatment was detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P-450 1A1 at the protein level. ELISAs showed that BA induces P-450 1A1 proteins 7.3-fold. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P-450 monooxygenase system which can be induced by P-450 inducers. In summary, we believe the presence of the cytochrome P-450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside

Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promotors of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM of cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase--mediated biotinylated--deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Induction of antitumor immunity by intracerebrally implanted rat C6 glioma cells genetically engineered to secrete cytokines.

To test whether cytokine gene therapy can be applied to an immunologically privileged site, such as the brain, we investigated antitumor immunity in the brain induced by cytokine-secreting glioma cells. Three cytokine genes, interleukin-2 (IL-2), interleukin-4 (IL-4), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were transduced into a rat C6 glioma cell line via a retroviral vector, S2. Rats intracerebrally (IC) implanted with the C6 cells genetically engineered to secrete the cytokines, especially GM-CSF, manifested significantly higher survival rates than those with C6 cells or with C6 cells bearing the control vector (p < 0.002). In vivo, C6 tumors bearing the cytokine genes grew more slowly than wild-type tumors at any time point, and eventually diminished within 6 weeks after tumor cell implantation. Histopathological and immunohistochemical studies revealed that different cytokines induced diverse immune reactions. In the IL-2 group, CD4+ and CD8+ T cells dominated from day 3 to week 4, but disappeared at week 6. Some granulocytes were noted between weeks 2 and 4. In the IL-4 group, eosinophils were noted from day 3 to week 4, and CD4+ and CD8+ T cells, as well as macrophages at week 2. At week 6, only residual levels of macrophages and CD8+ T cells remained. In the GM-CSF group, granulocytes appeared as early as day 1 post-IC tumor implantation, and macrophages at day 2. CD4+ and CD8+ T cells were found from day 3 to week 4. At week 6, only residual CD4+ T cells and macrophages remained. Long-lasting antitumor immunity was confirmed in all groups by rechallenging surviving rats with wild-type C6 cells in the brain 100 days after implanting cytokine gene-bearing C6 cells. In vivo depletion of GM-CSF by anti-GM-CSF antibody further confirmed that the immune reaction induced by GM-CSF-secreting tumor cells were mainly from the action of GM-CSF, rather than the immunogenicity of C6 cells.

Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas.

A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.

Radiation Response of Connexin43-Transfected Cells in Relation to the “Contact Effect”

Some cell lines grown for only two cell doublings as multicell spheroids develop a form of resistance to killing by ionizing radiation that has been called the “contact” effect. While our previous results have implicated a role for higher order chromatin structure in the contact effect, another possible explanation is the presence of intercellular gap junctions that might facilitate communication between cells grown as spheroids and thereby enhance the ability of cells to resist or recover from radiation damage. To examine the role of gap junctions in the contact effect, rat glioma C6 and mouse EMT6 cell lines were transfected with a gene encoding the gap junctional protein connexin43. While C6 glioma cells are deficient in gap junctional communication, cells from spheroids were nonetheless more resistant than monolayers to killing by ionizing radiation, and the contact effect was present to a similar extent in the three transfected clones. For mouse EMT6 cells, radiosensitivity was similar whether cells were grown as monolayers or spheroids. Transfection of EMT6 cells with connexin43 increased gap junctional communication but did not promote development of a contact effect. Tumor volume doubling time in SCID mice increased significantly for one transfected clone; however, doubling timein vitrowas also increased relative to the EMT6 parent. We conclude that extensive gap junctional communication is not a requirement for the increased radiation resistance observed when some cell lines are grown as spheroids.

Molecular and functional characterization of the promoter region of the mouse LDH/C gene: enhancer-assisted, Sp1-mediated transcriptional activation.

Molecular and functional studies of the LDH/C 5' upstream promoter elements were undertaken to elucidate the molecular mechanisms involved in temporal activation of LDH/C gene expression in differentiating germ cells. Ligation mediated-PCR (LM-PCR) gene walking techniques were exploited to isolate a 2.1 kb fragment of the mouse LDH/C 5' promoter region. DNA sequence analysis of this isolated genomic fragment indicated that the mouse LDH/C promoter contained TATA and CCAT boxes as well as a GC-box (Sp1-binding site) situated upstream from the transcription start site. PCR-based in vivo DNase I footprinting analysis of a 600 bp fragment of the proximal LDH/C promoter region (-524/+38) in isolated mouse pachytene spermatocytes identified a single footprint over the GC-box motif. Three DNase I hypersensitive sites were also detectable in vivo, in a region containing (CT)n(GA)n repeats upstream from the CCAT box domain. Functional characterization of the promoter region was carried out in a rat C6 glioma cell line and an SV40 transformed germ cell line (GC-1 spg) using wild-type and mutated LDH/C promoter CAT reporter constructs. These studies provide experimental evidence suggesting that transcriptional activation of the LDH/C promoter is regulated by enhancer-mediated coactivation of the Sp1 proteins bound to the GC-box motif footprinted in vivo in pachytene spermatocytes.

The role of tumour-derived mIL-4 on rat C6 glioma regression

Interleukin-4 (IL-4) has been demonstrated to possess anti-tumourigenic properties in vivo which is attributed to the infiltration of eosinophils in addition to an inhibition of tumour vascularisation. We have previously generated stable transfectants of C6 glioma cells that express mouse IL-4 (mIL-4) under a tetracycline-responsive promoter system, enabling us to apply tight regulatory control of mIL-4 expression in vivo. We have demonstrated that the subcutaneous implantation of mIL-4 expressing rat C6 glioma cell lines in nu/nu mice resulted in an inhibition of tumour growth. In this study, we have investigated the ability of mIL-4 to potentiate the regression of established subcutaneous rat C6 tumours in nu/nu mice. Induction of mIL-4 re-expression in established tumours did not cause a regression of tumour growth as determined by external tumour volume measurements. However, histological analysis revealed that mIL-4 re-expressing tumours were highly necrotic, were infiltrated by eosinophils and had a reduced level of vascularisation. These results suggest that mIL-4 can potentiate an anti-tumourigenic response in established C6 tumours in nu/nu mice. Furthermore, this study illustrates the need for detailed and defined tumour histopathology as tumour volume measurements alone can lead to inaccurate and misleading results.

Transduction of cytosine deaminase gene makes rat glioma cells highly sensitive to 5‐fluorocytosine

To investigate the potential use of E. coli cytosine deaminase (CD) gene instead of the commonly used HSV-TK gene in the gene therapy of brain tumors, we constructed a retrovirus vector carrying the CD gene. We then transduced a rat glioma cell line C6 with CD gene by the retrovirus vector. Transduction of the CD gene made C6 cells become highly sensitive to the anti-fungi drug 5-fluorocytosine (5FC). IC50 for 5FC was 6,000 μM in CD-negative cells, while it was 3 μM in Cd -positive cells. Mixed cellular assay showed that CD-positive cells had a strong “bystander effect” on CD-negative cells when exposed to 5FC. Significant anti-tumor effects were observed in nude mice bearing s.c. tumors derived from CD-positive cells when these animals were given 250 mg/kg 5FC twice a day for 20 consecutive days. A marked decrease in tumor weight occurred when a mixture containing 50% CD-positive and 50% CD-negative C6 cells was injected s.c., followed by 5FC treatment, suggesting the bystander effect in vivo. Concerning the pharmacokinetics of 5FC, especially its high oral bio-availability and good penetration into cerebrospinal fluid, we suppose that the combination of CD-gene transfer and 5FC oral administration may have potential use in the gene therapy of brain tumors. Int. J. Cancer 71:675-679, 1997. © 1997 Wiley-Liss, Inc.