Mouse Glioma Cells Research Articles

Antitumor activity of interleukin 12 against interleukin 2-transduced mouse glioma cells

We subcutaneously inoculated parental and glioma cells genetically engineered to express interleukin-2 (SR/IL-2) into syngeneic mice. The tumor growth of the transfectants was slower than that of the parental cells. We then stereotactically inoculated transfectants into the brains of mice. The survival of the mice injected with parental cells was shorter than that of the mice inoculated with transfectants. SR/IL-2 cells were inoculated subcutaneously into the flank of mice, after which rmIL-12 was administered intraperitoneally (i.p.). The resultant transient tumor growth was followed by regression. rmIL-12 or saline were then injected i.p. into mice that had been inoculated in the brain with SR/IL-2 cells. There was no significant difference in survival time between the treated and control groups.

Methylmercury-induced microtubule depolymerization leads to inhibition of tubulin synthesis.

Methylmercury (MeHg) specifically depolymerizes microtubules and inhibits cell proliferation in mouse glioma cells. The effect of microtubule depolymerization by MeHg on tubulin synthesis was studied. Tubulin synthesis analyzed by two-dimensional electrophoresis using [35S]methionine as a tracer was markedly inhibited in mouse glioma cells exposed to 5 x 10(-6)M MeHg for 3 hr, which completely depolymerized microtubules. Under this condition, density of the protein bands other than tubulin in autoradiogram remained unchanged on gradient urea-polyacrylamide gels. Furthermore, the decrease in tubulin mRNA level was relevant to that in tubulin synthesis, although actin mRNA levels remained unchanged. In addition, specific transcription rates of beta-tubulin genes appeared to be unaffected under the same experimental condition as above. Thus, it is concluded that the disruption of microtubules by MeHg resulted in the inhibition of the synthesis of tubulin itself through autoregulatory repression in post-transcriptional processes as in the case of colchicine treatment.

Gene delivery by HVJ-liposome in the experimental gene therapy of murine glioma.

We investigated the delivery of foreign genes into mouse glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. HVJ-liposomes, containing lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene-bearing plasmid DNA were applied in the meningeal gliomatosis (MG) mouse model system. Highly efficient delivery was observed in disseminated glioma cells, and 80% of MG mice expressing the HSVtk gene were cured by treatment with ganciclovir. These results suggest that this novel gene delivery system may be applicable for the in vivo gene therapy of human malignant glioma.

Usefulness of a mouse myelin basic protein promoter for gene therapy of malignant glioma: myelin basic protein promoter is strongly active in human malignant glioma cells.

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

Spatial and temporal regulation of the rat calmodulin gene III directed by a 877-base promoter and 103-base leader segment in the mature and embryonal central nervous system of transgenic mice.

Three non-allelic rat calmodulin (CaM) genes CaMI, CaMII and CaMIII, which share no homology in their 5'-upstream regions, are coordinately expressed in neurons of the central nervous system (CNS). Deletion analysis of the CaMIII promoter showed that the upstream segments longer than 700 bases functioned as efficient promoters, and that the sequence from -133 to -65 was required for the activity of house-keeping type promoter in transient expression assays on a mouse glioma cell line C6. However, the transient expression seemed not to be cell type specific. To determine the temporal and spatial specificity of the promoter function, we produced transgenic mice carrying a fusion gene of the CaMIII segment from -877 to +103 and the lacZ reporter gene. In CNS of the adult transgenic mice, the localization of transgene expression was similar to that of endogenous CaMIII transcripts analyzed by in situ hybridization. The transgene was expressed prominently in pyramidal cells of the cerebral neocortex and the hippocampal regions CA1 to CA3, in Purkinje cells of the cerebellar cortex, and in neurons of the spinal cord, and moderately in granule cells of the dentate gyrus and the cerebellar cortex. In the developing CNS, the overall profiles of neuron-specific expression were also similar for both transgene and endogenous CaMIII that were expressed in the mantle layer and the dorsal root ganglia of the embryonal spinal cord. These results indicated that the neuron-specific expression of rat CaMIII was directed by this 877-base promoter sequence. The CaMIII segment used for the promoter of transgene contained a 29-bp sequence at -410, namely H3, which was conserved in the upstream regions of vertebrate CaMII and CaMIII. H3 seemed to play a pivotal role in the temporal and spatial expression of transgene in CNS, although the deletion of H3 did not decrease CAT activity in the transient expression. The transgene expression was not observed in the external granular cells of the developing cerebellum and in some neurons of the embryonic sensory ganglia in which the endogenous CaMIII was obviously expressed. Therefore, the other cis-acting element(s) located outside of this 877-bp segment seemed to be required for the temporal regulation of CaMIII in certain rudimentary neurons.

Heterogeneity of Protein Kinase C Activity in Human U-373 and G-26 Mice Glioma Cells

Protein kinase C (PKC) enzyme activity in a mouse glioma cell line G-26 and a human glioma cell line U-373 were compared at similar cell confluency in-vitro to establish if a G-26 in-vivo mouse model would be useful to examine the role of PKC inhibitors in controlling human glioma growth in-vivo. Original crude cytosolic and membrane PKC fractions of both mouse glioma G-26 and human glioma U-373 cells did not display significant PKC activity compared to partially purified PKC. Partial purification of mouse glioma G-26 and U-373 cytosolic and membrane fractions showed different cytosolic and membrane PKC activity profiles. Total PKC activity was higher (ρ = 0.0001) in human glioma U-373 (7840 picomoles/mg/min) than in mouse glioma G-26 cells (2890 picomoles/mg/min). Thus, results from trials using nude mice human glioma xenografts may be more valid than those obtained from a G-26 in-vivo mouse model for studying the effects of therapeutic drugs on PKC isozymes.

Selective expression of foreign genes in glioma cells: use of the mouse myelin basic protein gene promoter to direct toxic gene expression.

We have previously demonstrated that retrovirus-mediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244-1247, 1992). This retrovirus vector contains the Escherichia coli. beta-galactosidase (beta-gal) gene as a marker for integration of the lacZ gene, which is controlled by the SV40 early promoter. We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial-specific promoters, including the 2.5 kb 5' flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5' flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5' flanking region of the myelin proteolipid protein (PLP) gene. Psi-2 packaging cells were transfected with each retrovirus vector (GFAP promoter-, MBP promoter-, and PLP promoter-lacZ) and the infectious virus particles were recovered from the supernatants. Blue staining for beta-gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAG vector. On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene-bearing retrovirus controlled by glial-specific promoters. The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene. Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of MHC Induction by Mycoplasma fermentans (Incognitus Strain)

Mycoplasma fermentans (incognitus strain) is a recently identified new human pathogen and suspected cofactor in acquired immune deficiency syndrome. Because this organism appears to exert strong immunosuppressive properties of its own, we decided to investigate whether it was capable of inducing MHC class II expression, as we have observed for other species of mycoplasma. In this report we demonstrate that M. fermentans (incognitus strain) is capable of producing factors that increase MHC class II expression as well as MHC class I expression on the myelomonocytic cell line, WEHI-3 cells, We also present data showing that these mycoplasmal factors induce small, although significant, increases in MHC class I and II antigens on a mouse glioma cell line, G26-20, and MHC class II expression on the human monocyte cell lines, U-937 and HL-60. Using nuclear run-on analysis, we show that the mycoplasma-induced increase in MHC expression is at least partially due to an increase in transcription of the MHC genes. Furthermore, we show that the factor that mediates this activity is sensitive to protease treatment, indicating that it is, at least in part, protein. These results demonstrate that M. fermentans (incognitus strain) is capable of modulating the expression of immunologically important MHC genes in both murine and human cell lines, which may prove to be an important factor in the pathogenesis of this organism.

Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain.

A murine model for meningeal metastasis of malignant glioma was developed to study selective gene transfer into tumor cells and to establish a reliable means of determining the rate of tumor cell infection. A murine ecotropic retroviral vector was created in which the Escherichia coliβ‐galactosidase gene served as a marker for gene expression from the integrated retrovirus. This retrovirus exhibited a high rate of infectivity in RSV‐M mouse glioma cells in vitro. The recombinant retrovirus was injected directly into the cisterna magna of the mice. Staining of β‐galactosidase showed that the rate of gene integration was high in the disseminated glioma cells. These results suggest the possibility of retrovirus‐mediated gene therapy for meningeal dissemination of malignant glioma.

Chromosome aberrations of human small cell lung cancer induced by a new 111In-bleomycin complex.

A new 111Indium labeled bleomycin complex (111In-BLMC) was prepared and found to be effective for tumor imaging and therapy both in mouse glioma and human small cell lung cancer (SCLC) cells. Chromosome aberrations were studied in human SCLC cells to explore its mechanisms of killing cancer cells. SCLC cells (N417) were exposed to 111In-BLMC, BLM, or 111InCl3 (for control) for 1 hour, treated with colcemid, and chromosomal changes were analyzed. A dramatic increase in chromatic gaps, breaks, chromosome breaks, double minutes, rings, triradii, quadriradii, and chromosome stickiness were observed in the cells treated by 111In-BLMC compared to BLM or 111InCl3. These results indicated that 111In-BLMC has therapeutic potential for combination chemo-radiotherapy of cancer (e.g., by Auger electrons and local energy deposition).

Expression and modulation of major histocompatibility antigens on murine primary brain tumor in vitro

Lysis of tumor cells by activated cytotoxic lymphocytes requires their recognition of antigens associated with major histocompatibility complex molecules. The authors studied the constitutive expression of Class I and Class II major histocompatibility complex antigens on mouse brain-tumor cells and the capacity of different cytokines and cytokine combinations to alter this expression in vitro. Cells from the murine glioma 26 (GL26), glioma 261 (GL261), and ependymoblastoma A (EpA) cell lines were established in monolayer culture and treated for 48 hours with either alpha interferon, gamma interferon, tumor necrosis factor alpha, tumor necrosis factor alpha plus gamma interferon, or interleukin-2. They were then analyzed by flow cytometry for baseline and cytokine-altered major histocompatibility complex expression. All cell lines had a similar constitutive major histocompatibility complex pattern with low Class I antigen expression and no detectable Class II antigen expression. Alpha interferon substantially induced and up-regulated Class I antigen expression, but had no effect on Class II antigen expression. Gamma interferon also stimulated up-regulation of Class I antigen expression, generally doubling the anti-Class I antigen fluorescence of treated cells. Its effect on Class II antigen expression was more extensive. In the GL26 and GL261 cell lines the expression of Class II antigen determinants increased to 12 x and 14 x control values and as many as 75% of cells that had no detectable constitutive expression of Class II antigen expressed this antigen after priming with gamma interferon. The addition of tumor necrosis factor alpha to gamma interferon further increased Class II antigen expression on EpA tumor cells only. Interleukin-2 and tumor necrosis factor alpha alone had no effect on Class I or Class II antigen expression of any cell lines. It is concluded that Class I and Class II antigen expression in mouse glioma cell lines is induced and enhanced after treatment with certain cytokines in vitro. Use of these cell lines to create in situ primary brain tumors in C57BL/6 mice should provide an excellent animal system to study major histocompatibility complex modulation in brain tumor cells and to examine the potential impact of major histocompatibility complex up-regulation on the response of brain tumors to immunotherapy.

Characterization of nuclear T3 receptors in human neuroblastoma cells SH-SY5Y: Effect of differentiation with sodium butyrate and nerve growth factor

Nuclear receptors for the thyroid hormone triiodothyronine (T3) have been identified in vivo in brain tissues and in vitro in mouse and rat neuroblastoma and glioma cells. The present study characterizes nuclear T3 receptors in human neuroblastoma SH-SY5Y cells and compares their level before and after differentiation. Undifferentiated cells, grown in DME/HAM F-12 medium supplemented with 10% fetal calf serum, show an abundant single type of nuclear receptor, indicated by a straight Scatchard plot, with a Kd of 0.11 nmol/l. After treatment with sodium butyrate (0.5 mM for 4 days) or NGF (2 nM for 6 days), the cells showed neuronal-like patterns (extension of neurites, slowing of growth, increased tyrosine hydroxylase activity), with a decrease in the number of nuclear T3 receptors. As sodium butyrate and NGF treatments differentiate neuroblastoma SH-SY5Y cells, these data suggest a down-regulation of T3 receptors with cell maturation.

Mechanism of cytotoxicity of methylmercury

Mechanism of methylmercury cytotoxicity was investigated with special reference to its preferential action on microtubules and protein biosynthesis in cultured cells. The tubulin synthesis analyzed by autoradiography of two-dimensional electropherogram using 35S-methionine was inhibited by 50-70% in mouse glioma cells exposed to 5 x 10(-6) M methylmercury for 3 h, which almost completely depolymerized microtubules. Total protein synthesis monitored by incorporation of labeled methionine into acid insoluble fraction was decreased slightly but significantly and the protein bands other than tubulin on gradient urea-PAGE gel appeared to remain unchanged under the experimental condition used. These results suggest that the inhibition of protein synthesis observed on exposure to methylmercury can be ascribed, at least partly, to a possible autoregulatory depression in tubulin synthesis owing to the increase in the pool of tubulin subunits resulted from microtubule depolymerization by methylmercury.

Growth-inhibitory effect of prostaglandin D2 on mouse glioma cells.

The effects of prostaglandin D2 (PGD2) on the growth of mouse malignant glioma cells were studied in vitro and in vivo. The in vitro studies consisted of various concentrations of prostaglandins (PG's) being added to cultures of mouse glioma cells. At concentrations above 2.5 micrograms/ml, PGD2 strongly inhibited the proliferation of glioma cells, whereas PGE2 had no effect at the same value. Exposure to 5.0 micrograms/ml PGD2 for more than 2 hours resulted in inhibition of glioma cell proliferation. This growth-inhibitory effect of PGD2 was related to the inhibition of DNA synthesis of the cells. The in vivo studies were performed with a subcutaneously transplanted mouse glioma model. Injection of 0.5 mg/kg PGD2 into the tumor was more effective than the same concentration given by intraperitoneal injection. In mice with intracranially transplanted glioma, daily intraperitoneal injection of 0.5 mg/kg PGD2 had no significant effect on survival time.

Effects of methylmercury and some metal ions on microtubule networks in mouse glioma cells and in vitro tubulin polymerization

Mercury compounds and some other metal ions were investigated with respect to their effect on in vitro tubulin polymerization and on cellular microtubules in mouse glioma. In vitro tubulin polymerization was completely inhibited by 2.5 × 10 −5 m Hg 2+, 5 × 10 −5 m CH 3Hg +, 2 × 10 −4 m Cr 3+, 2.5 × 10 −4 m Cu 2+, and 5 × 10 −4 m Cd 2+. Zn 2+ did not affect the polymerization up to 5 × 10 −4 m. Indirect immunofluorescence study with rabbit antiporcine tubulin antibody revealed that methylmercury disrupted the microtubule network at an early stage of growth inhibition. On the other hand, in the presence of Cd 2+, Ci 2+, and Cr 3+ at their growth inhibitory concentrations, no effects on microtubule networks were observed for the first 1 hr. These results indicate that only methylmercury affects cellular microtubules, while other ions seem to interfere with other sites in the cells, although these ions showed the ability to depress in vitro tubulin polymerization.

Local immunotherapy of malignant brain tumors. Evaluation of intratumoral injection of BCG and a Streptococcus pyogenes preparation

The anti-tumor effect of local immunotherapy using BCG and Picibanil, a Streptococcus pyogenes preparation, was evaluated in experimental and human malignant brain tumors. BCG and Picibanil produced an identical inflammatory cell reaction in the C3HHe mouse brain after intracerebral injection. BCG had no direct cytocidal effect on Rous sarcoma virus-induced malignant glioma cells of the mouse in vitro. A mixture of the mouse glioma cells and BCG was inoculated into the subcutaneous tissue and brain of the syngeneic mice. The development and growth of subcutaneous and brain tumors were inhibited remarkably by the addition of 100-1, 000 μg of BCG to 2×105 subcutaneously transplanted tumor cells, and by the addition of 50-500 μg of BCG to 1×104 intracerebrally transplanted tumor cells. Intratumoral injection of 5 mg of BCG showed a significant suppression of the growth of subcutaneous tumors which could not be obtained by the systemic administration of the same dose of BCG. The enhancement of tumor-specific immunity was obtained in this mouse-tumor system by the repeated immunization of a mixture of irradiated tumor cells and BCG. Thirteen patients with malignant brain tumors, 12 anaplastic gliomas and one metastatic tumor, received repeated intratumoral injections of Picibanil through one to two intratumorally implanted tubes. All patients tolerated this therapy, and the morbidity rate was acceptable. Significant tumor regression was noted on CT scans in 6 of 11 patients for whom scanning was performed. Histologic examinations of the post-therapy specimens obtained from 9 patients revealed that inflammatory cell reactions were evoked in all of the tumors, although the extent of the inflammatory reactions varied from tumor to tumor and was mostly localized to an area surrounding the intratumoral tubes. Combination therapy of local Picibanil injection and systemic administration of ACNU as maintenance immunochemotherapy was performed in another series of anaplastic glioma patients. Among the 10 patients who were followed for more than 2 years after treatment, 5 survived more than 2 years and 4 obtained long-term survivals of 71, 58, 44 and 43 months postoperatively. In conclusion, local immunotherapy is worth employing as maintenance immunochemotherapy for anaplastic glioma patients.

<b>Isolation of glial fibrillary acidic protein-positive clonal mouse glioma cell line</b>

A new clonal astrocytic cell line (F6) was isolated from a Rous sarcoma virus-induced mouse brain tumor cells. This clone expresses glial fibrillary acidic protein (GFAP) antigen and simultaneously possesses many intermediate filaments in nearly 100% ofcells. The arrangement ofthe intermediate filaments in the F6 cells is in good accordance with the fibrillar staining pattern of GFAP. These findings suggest that the F6 clone has the well-developed astrocytic cytoskeleton. The clone would be of value in studying glial intermediate filaments and other biological characteristics of astrocytes.

Stimulation of DNA synthesis and pyrimidine deoxyribonucleoside transport systems in mouse glioma and mouse neuroblastoma cells by inorganic mercury

Biochemical studies on effects of methylmercury and inorganic mercury on mouse glioma and neuroblastoma cells were carried out. Inorganic mercury accelerated DNA synthesis of mouse glioma cells at 10 −6 to 10 −5 m specifically in the middle log phase of the growth. Inorganic mercury facilitated the thymidine and deoxycytidine transports within a narrow range of concentration (1 to 2 × 10 −5 m), but no stimulation of transport was observed in the cases of purine deoxyribonucleosides. Inorganic mercury markedly inhibited both DNA synthesis and the transport systems of the precursors above 5 × 10 −5 m. On the other hand, methylmercury showed hardly any accelerating effects and markedly inhibited both the synthesis of DNA and the precursors' transport above 5 × 10 −5 m. In the case of mouse neuroblastoma cells, inorganic mercury stimulated DNA synthesis at 1 to 5 × 10 −6 m and also thymidine transport system at 1 to 2 × 10 −5 m.

Regulation of glycosphingolipid metabolism in mouse neuroblastoma and glioma cell lines. Comparison of glioma (oligodendroglioma-like) with neutroblastoma cell lines.

Regulation of glycosphingolipid metabolism in mouse neuroblastoma and glioma cell lines. Comparison of glioma (oligodendroglioma-like) with neutroblastoma cell lines.

Effects of methylmercury on mitotic mouse glioma cells

The effects of methylmercury on cultured mammalian cells were studied by light and electron microscopy. At 5 × 10 −6 m concentration methylmercury depressed the proliferation of mouse glioma cells almost completely and increased the mitotic indices. In the electron micrographs of the cells treated with 5 × 10 −6 m methylmercury for 4 hr, microtubules as mitotic spindle fiber were absent and chromosomes were scattered in a disorderly fashion in the cytoplasm. From these observations we concluded that exposure to methylmercury inhibits the cell mitosis by blocking the polymerization of tubulin to microtubules and results in the accumulation of the cells on the way of mitosis. Changes in behaviors of other mitotic apparatus and of fibrillar structures other than microtubules are also described.