tetracycline and Glioma

Topics: Adult; Aged; Angiography; Animals; Blood Circulation; Brain Neoplasms; Carcinogens; Cats; Chloroquine; Choroiditis; Dogs; Eye Diseases; Female; Fluorescence; Glioma; Haplorhini; Hemorrhage; Humans; Mestranol; Microscopy, Electron; Neuritis; Norethynodrel; Optic Nerve; Oxygen Consumption; Papilledema; Polyarteritis Nodosa; Rabbits; Retina; Retinal Detachment; Retinal Vessels; Tetracycline

Topics: Amphotericin B; Anti-Infective Agents; Biopsy; Brain Neoplasms; Central Nervous System Infections; Diagnosis, Differential; Diplopia; Face; Female; Gait Disorders, Neurologic; Glioma; Humans; Immunocompetence; Infections; Magnetic Resonance Imaging; Middle Aged; Neurosurgical Procedures; Paresthesia; Prototheca; Taste Disorders; Tetracycline

Nitric oxide (NO) is a free radical signalling molecule involved in various physiological and pathological processes, including cancer. Both tumouricidal and tumour promoting effects have been attributed to NO, making its role in cancer biology controversial and unclear. To investigate the specific role of tumour-derived NO in vascular development, C6 glioma cells were genetically modified to include a doxycycline regulated gene expression system that controls the expression of an antisense RNA to inducible nitric oxide synthase (iNOS) to manipulate endogenous iNOS expression. Xenografts of these cells were propagated in the presence or absence of doxycycline. Susceptibility magnetic resonance imaging (MRI), initially with a carbogen (95% O2/5% CO2) breathing challenge and subsequently an intravascular blood pool contrast agent, was used to assess haemodynamic vasculature (ΔR2*) and fractional blood volume (fBV), and correlated with histopathological assessment of tumour vascular density, maturation and function. Inhibition of NO production in C6 gliomas led to significant growth delay and inhibition of vessel maturation. Parametric fBV maps were used to identify vascularised regions from which the carbogen-induced ΔR2* was measured and found to be positively correlated with vessel maturation, quantified ex vivo using fluorescence microscopy for endothelial and perivascular cell staining. These data suggest that tumour-derived iNOS is an important mediator of tumour growth and vessel maturation, hence a promising target for anti-vascular cancer therapies. The combination of ΔR2* response to carbogen and fBV MRI can provide a marker of tumour vessel maturation that could be applied to non-invasively monitor treatment response to iNOS inhibitors.

Topics: Animals; Cell Line, Tumor; Contrast Media; Free Radicals; Gene Expression Regulation, Neoplastic; Glioma; Humans; Magnetic Resonance Imaging; Mice; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Synthase Type II; Tetracycline; Xenograft Model Antitumor Assays

Dendritic cell factor 1 plays important roles in neural stem cells differentiation and in glioma cells proliferation, migration, and invasion. Here, we used a tetracycline—inducible system that regulates the expression of Dendritic cell factor 1 in glioma cells. We constructed two tet—inducible vectors, pTRE—EGFP—DCF1 and pTRE—LJM1—DCF1, by modifying the promoter PCMV. In the absence of tetracycline or doxycycline, the expression of Dendritic cell factor 1 in cells co—transfected with pTRE—EGFP—DCF1 or pTRE—LJM1—EGFP—DCF1 and ptTS—Neo was suppressed through binding of the tetracyline—controlled transcriptional suppressor to tetracycline response element, and the suppression was released by the addition of doxycycline. Our work has laid foundations for potential clinical application of cancer therapy in realizing artificial regulation of gene.

Topics: Anti-Bacterial Agents; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dendritic Cells; Doxycycline; Gene Expression Regulation, Neoplastic; Glioma; HEK293 Cells; Humans; Membrane Proteins; Neoplasm Invasiveness; Nerve Tissue Proteins; Neural Stem Cells; Promoter Regions, Genetic; Protein Binding; Response Elements; Tetracycline

Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the "OFF" state, and expression should quickly reach therapeutic levels in the "ON" state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA(2)S-M2 inducer and tTS(Kid) silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo.

Topics: Adenoviridae; ADP Ribose Transferases; Anti-Bacterial Agents; Bacterial Toxins; Blotting, Southern; Cells, Cultured; Diphtheria Toxin; Doxycycline; Exotoxins; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Glioma; Humans; Pseudomonas aeruginosa Exotoxin A; Tetracycline; Transgenes; Virulence Factors

The heavy chain of murine ferritin, an iron storage molecule with ferroxidase activity, was developed as a novel endogenous reporter for the detection of gene expression by magnetic resonance imaging (MRI). Expression of both enhanced green fluorescent protein (EGFP) and influenza hemagglutinin (HA)-tagged ferritin were tightly coregulated by tetracycline (TET), using a bidirectional expression vector. C6 cells stably expressing a TET-EGFP-HA-ferritin construct enabled the dynamic detection of TET-regulated gene expression by MRI, followed by independent validation using fluorescence microscopy and histology. MR relaxation rates were significantly elevated both in vitro and in vivo on TET withdrawal, and were consistent with induced expression of ferritin and increase in intracellular iron content. Hence, overexpression of ferritin was sufficient to trigger cellular response, augmenting iron uptake to a degree detectable by MRI. Application of this novel MR reporter gene that generates significant contrast in the absence of exogenously administered substrates opens new possibilities for noninvasive molecular imaging of gene expression by MRI.

Topics: Animals; Anti-Bacterial Agents; Brain Neoplasms; Cell Proliferation; Female; Ferritins; Gene Expression; Genes, Reporter; Glioma; Green Fluorescent Proteins; Hemagglutinin Glycoproteins, Influenza Virus; In Vitro Techniques; Iron; Magnetic Resonance Imaging; Mice; Mice, Nude; Microscopy, Fluorescence; Tetracycline; Transfection; Tumor Cells, Cultured

LRRC4 is a novel relatively specific gene, which displays significant down-regulation in primary brain tumor biopsies and has the potential to suppress brain tumor growth. In this study, we investigated the growth inhibitory effect of LRRC4 on tumorigencity in vivo and on cell proliferation in vitro by a tetracycline-inducible expression system. Results showed that LRRC4 significantly reduced the growth and malignant grade of xenografts arising from glioblastoma U251MG cells. Cell proliferation was markedly inhibited after U251MG Tet-on-LRRC4 cell induction with doxycycline. Flow cytometry and Western blot analysis demonstrated that LRRC4 mediated a delay of the cell cycle in late G1, possibly through up-regulating the expressions of p21Waf1/cip1 and p27Kip1 and down-regulating the expressions of cyclin-dependent kinase 2, retinoblastoma protein and epidermal growth factor receptors. Together, these findings provide clues to the function of LRRC4 as a negative regulator of cell growth and underscore a link between the above-mentioned cyclins, cyclin-associated molecules and tumorigenicity.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Genes, Tumor Suppressor; Glioma; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nerve Tissue Proteins; Tetracycline; Transfection

We have investigated growth and vascularization of malignant glioma in mice upon conditional inhibition of fibroblast growth factor (FGF) receptor activity. C6 rat glioma cells were transfected with a dominant-negative fibroblast growth factor receptor-2 (FGFR2-DN) cDNA under the control of a tetracycline-regulated expression promoter (tet off) and implanted in the brain of immunodeficient mice. Magnetic resonance imaging analysis showed a significant decrease in tumor growth 14 days after implantation when FGFR2-DN was expressed compared to control. This size difference disappeared after 20 days. However, after 20 days, tumor and endothelial cells apoptosis were higher in the FGFR2-DN group and consequently angiogenesis was decreased whereas tumor cells were similarly associated with blood vessels at the tumor periphery. Pericyte coverage was not different between the two groups but a higher amount of pericytes not associated with vessels was found in the FGFR2-DN expressing group. This demonstrates, that conditional expression of inhibitor of FGF receptor activity in gliomas implanted in the brain of immunodeficient mice can be achieved efficiently, and that FGFs are major players in glioma development and in glioma angiogenesis.

Topics: Animals; Apoptosis; Brain; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation; Genetic Vectors; Glioma; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Rats; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Tetracycline; Transfection; Vascular Endothelial Growth Factor A

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.

Topics: Adenoviridae; Animals; Blotting, Western; Brain; Cell Line; Doxycycline; Gene Expression Regulation; Genetic Vectors; Glioma; Kinetics; Reproducibility of Results; Tetracycline; Transcription, Genetic; Transgenes; Tumor Cells, Cultured

Conditional expression systems for 4-repeat wild-type (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 x g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.

Topics: Chromosomes, Human, Pair 17; Dementia; Gene Expression Regulation; Glioma; Humans; Immunoblotting; Immunohistochemistry; Inclusion Bodies; Kinetics; Microscopy, Fluorescence; Microtubule-Associated Proteins; Mutation; Parkinson Disease; Phosphorylation; tau Proteins; Tetracycline; Transfection; Tumor Cells, Cultured

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Cell Division; DNA, Complementary; Enzyme Inhibitors; ErbB Receptors; Glioma; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; RNA; RNA, Messenger; Tetracycline; Time Factors; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine; Tyrphostins

We describe here the oncostatin M (OSM)-dependent inhibition of in vivo tumour formation after intracerebral inoculation of glioblastoma cells in mice. We generated human glioblastoma cells transfected with the OSM gene under the control of a tetracycline-response promoter. Upon removal of tetracycline from the medium, cells exhibited a differentiated cell morphology, while proliferation was significantly inhibited. After implantation of these cells into nude mice brains, large tumours developed in animals lacking OSM expression, whereas no tumour formation was observed in mice with induced OSM expression. Our results suggest that OSM exerts pronounced antitumorigenic effects on glioblastoma cells in vivo and provide arguments for a therapeutic application of OSM in humans.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Division; Female; Glioma; Humans; Mice; Mice, Nude; Oncostatin M; Peptides; Promoter Regions, Genetic; Recombinant Proteins; Tetracycline; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

We undertook a series of systematic studies to address the role of fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) activity in tumor growth and angiogenesis. We expressed dominant-negative FGFR2 (FGFR2-DN) or FGFR1 (FGFR1-DN) in glioma C6 cells by using constitutive or tetracycline-regulated expression systems. Anchorage-dependent or independent growth was inhibited in FGFR-DN-expressing cells. Tumor development after xenografting FGFR-DN-expressing cells in immunodeficient mice or after transplantation in rat brain was strongly inhibited. Quantification of microvessels demonstrated a significant decrease in vessel density in tumors derived from FGFR-DN-expressing cells. Furthermore, in a rabbit corneal assay, the angiogenic response after implantation of FGFR-DN-expressing cells was decreased. In tumors expressing FGFR-DN, vascular endothelial growth factor expression was strongly inhibited as compared with control tumor. These results indicate that inhibition of FGF activity may constitute a dominant therapeutic strategy in the treatment of FGF-producing cerebral malignancies and may disrupt both angiogenesis-dependent and -independent signals required for glioma growth and invasion.

Topics: Animals; Brain Neoplasms; Cell Division; Endothelial Growth Factors; Fibroblast Growth Factor 2; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Glioma; Lymphokines; Male; Mice; Neovascularization, Pathologic; Phenotype; Proto-Oncogene Proteins; Rabbits; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Tetracycline; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transcription control systems enable experimental regulation of transgene expression in eukaryotic cells by application of specific repressors or inducers. Currently used inducible systems include tetracycline (tet-off), dimerizer and ecdysone systems. While numerous studies have utilized a single system, their comparative performance under identical conditions remains unclear. We therefore compared the efficiency of these three systems in C6 glioma cells by using transient transfection and the lacZ reporter gene. Highest induced activity was found with the ecdysone system, followed by tet-off and dimerizer systems. Both lowest repressed activity and highest regulation were revealed with the dimerizer system, followed by ecdysone and tet-off systems. Our data suggest that the most appropriate system depends on the experimental procedures, the application and the gene to be regulated.

Topics: Animals; Dimerization; Ecdysone; Ecdysterone; Gene Expression Regulation, Neoplastic; Glioma; Protein Synthesis Inhibitors; Rats; Tetracycline; Transfection; Tumor Cells, Cultured

Features that distinguish tumor vasculatures from normal blood vessels are sought to enable the destruction of preformed tumor vessels. We show that blood vessels in both a xenografted tumor and primary human tumors contain a sizable fraction of immature blood vessels that have not yet recruited periendothelial cells. These immature vessels are selectively obliterated as a consequence of vascular endothelial growth factor (VEGF) withdrawal. In a xenografted glioma, the selective vulnerability of immature vessels to VEGF loss was demonstrated by downregulating VEGF transgene expression using a tetracycline-regulated expression system. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy. VEGF loss led, in turn, to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that the unique dependence on VEGF of blood vessels lacking periendothelial cells can be exploited to reduce an existing tumor vasculature.

Topics: Androgens; Animals; Apoptosis; Blood Vessels; Down-Regulation; Endothelial Growth Factors; Gene Expression Regulation, Neoplastic; Glioma; Humans; In Situ Nick-End Labeling; Lymphokines; Male; Mice; Mice, Nude; Neoplasms, Experimental; Prostatic Neoplasms; RNA, Messenger; Tetracycline; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Homozygous chromosome 9p deletions in gliomas commonly include the CDKN2A and CDKN2B genes, which code for the structurally highly homologous cdk inhibitors/tumor suppressors p16 and p15, respectively. Alternative splicing of the CDKN2A gene results in the expression of p14(ARF). Interestingly, not only p16 and p15, but also the structurally unrelated p14(ARF) appear to function as negative cell cycle regulators. Concerted inactivation of p16, p15 and p14(ARF) could be demonstrated in seven of nine glioblastoma cell lines. Strong suppression of tumorigenicity after transfection with p16 and p15 alone or in combination was seen in cell lines containing neither endogenous p16 nor p15 but functional pRB. Significantly weaker growth suppression was observed in tumors either retaining expression of both p16 and p15 or p15 only. p14(ARF) proved to be a potent tumor suppressor in the presence of wild-type p53, while mutant p53 substantially reduced growth inhibition by p14(ARF). No differences between p16 and p15 effects could be observed, suggesting a largely overlapping function of p16 and p15. To facilitate further research into p16/p15 effects, three cell lines with conditional, tetracycline-controlled p16 expression were established. Reversible growth suppression mediated by p16 was observed in these models. Combined inactivation of CDKN2A and CDKN2B, i.e., loss of both p16 and p15 as well as p14(ARF), results in disruption of two major growth control pathways involving pRB and p53 in malignant gliomas. Therefore, homozygous co-deletions of CDKN2A and CDKN2B rather than mutations targeting individual transcripts are frequently selected for in these tumors.

Topics: Base Sequence; Brain Neoplasms; DNA, Neoplasm; Gene Silencing; Genes, p16; Glioma; Humans; Molecular Sequence Data; Mutation; Proteins; Tetracycline; Tumor Cells, Cultured; Tumor Suppressor Protein p14ARF

The principle hurdles for gene therapy are selectivity and efficacy. Toward that end, we constructed an adenovirus gene delivery system to enable robust, glial-specific, and repressible ectopic expression. A replication-incompetent (E1-deleted) adenovirus 5 vector was modified by the addition of three tandem repeats of a 300-bp fragment enhancer region of the glial fibrillary acidic protein gene coupled to a minimal promoter sequence from human cytomegalovirus to drive a tetracycline-controlled transactivator. Using beta-galactosidase as a reporter gene, we demonstrated high level expression in cells of glial origin (including cell lines derived from glioblastoma multiforme) but no detectable expression in nonglial cells (neuroblastoma or fibroblasts). Furthermore, expression was tightly regulated by anhydrous tetracycline. To our knowledge, this is the first gene therapy delivery system that is glial specific and which also allows for repression of ectopic gene expression.

Topics: Adenoviridae; Anti-Bacterial Agents; beta-Galactosidase; Brain Neoplasms; Defective Viruses; Genes, Reporter; Genetic Therapy; Genetic Vectors; Glial Fibrillary Acidic Protein; Glioma; Humans; Sensitivity and Specificity; Tetracycline; Tumor Cells, Cultured

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.

Topics: 3T3 Cells; Animals; Base Sequence; Blotting, Western; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Size; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Glioma; Humans; Immunohistochemistry; Mice; Models, Biological; Oncogene Proteins; Proto-Oncogene Proteins; Retinoblastoma-Binding Protein 1; Tetracycline; Time Factors; Transcription Factor DP1; Transcription Factors; Transcriptional Activation; Tumor Suppressor Protein p53

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously "on," tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.

Topics: Animals; Cell Line; Endothelial Growth Factors; Gene Expression Regulation, Neoplastic; Glioma; Hemangioblastoma; Lymphokines; Mice; Mice, Nude; Neoplasms, Experimental; Neovascularization, Pathologic; Protein Synthesis Inhibitors; Tetracycline; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

p16INK4A is a G1-specific cell cycle inhibitor which maps to human chromosome 9p21, a region frequently mutated or deleted in cancer cell lines and primary tumors. In glioblastomas the frequency of homozygous deletions is 40-70% making it one of the most common mutations in this tumor type. We have analysed the significance of the loss of this gene in gliomas by introducing the cDNA for p16INK4A into the human glioma cell line U-1242 MG which has a deleted CDKN2 locus. We used the tetracycline repressible vector system and obtained two stably transfected clones that expressed p16INK4A upon induction. p16INK4A expression caused a G1 arrest and enlargement of the cells similar to that of senescent cells. When staining for Senescence-Associated beta-galactosidase activity, described to be specific for senescent cells, we could show that the enlarged cells specifically gave a positive staining reaction. This senescence phenotype was dependent on the continuous expression of p16INK4A since it was reversed upon reintroduction of tetracycline suppression. Thus, the induced expression of p16INK4A in these glioma cells reverted their immortal phenotype and caused an immediate cellular senescence.

Topics: Apoptosis; beta-Galactosidase; Carrier Proteins; Cell Division; Cellular Senescence; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; G1 Phase; Gene Deletion; Gene Expression Regulation, Neoplastic; Genetic Vectors; Glioma; Homozygote; Humans; Phenotype; Proto-Oncogene Proteins; Retinoblastoma Protein; Tetracycline; Vimentin

Interleukin-4 (IL-4) has been demonstrated to possess anti-tumourigenic properties in vivo which is initially attributed to the infiltration of eosinophils proposed to occur by IL-4 binding to its receptors on endothelial cells, thereby mediating eosinophil adhesion. We have investigated whether the binding of IL-4 to receptors on endothelial cells could elicit other biological responses which may also play a role in tumour inhibition, such as angiogenesis. We have demonstrated that mouse IL-4 (mIL-4) down-regulates the expression of one of the receptors for VEGF, VEGF-R2, on endothelial cells in vitro. By generating stable transfectants of C6 glioma cells that express mIL-4 under a tetracycline-responsive promoter system, we were able to apply tight regulatory control of mIL-4 expression in vivo. Subcutaneous implantation of mIL-4/C6 cell lines in nu/nu mice revealed that tumour growth is inhibited by mIL-4 expression. mIL-4-expressing tumours were demonstrated to have a reduced level of vascularisation compared with controls, in addition to a high degree of eosinophil infiltration. Our results suggest that mIL-4 has bimodal biological roles in potentiating tumour inhibition in athymic mice: the suppression of angiogenesis and the augmentation of the host local immune response.

Topics: Animals; Brain Neoplasms; Cell Division; Cloning, Molecular; DNA, Complementary; Endothelial Growth Factors; Endothelium; Eosinophils; Female; Genetic Vectors; Glioma; Interleukin-4; Lymphokines; Male; Mice; Mice, Nude; Necrosis; Neovascularization, Pathologic; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Tetracycline; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Diffuse invasion of brain tissue by single tumor cells is a characteristic feature of gliomas and a major reason why these tumors cannot be completely resected. The molecular basis of brain invasion is poorly understood. We regulated the expression of beta 1 integrins, the major group of extracellular matrix receptors, in astrocytic tumor cells by using a tetracycline-dependent transcription control system. Rat C6 glioma cells were stably transfected with (a) the tetracycline-controlled transactivator (tTA) gene, (b) antisense beta 1 cDNA under the control of a tTA/tetracycline-responsive promoter, and (c) the beta-galactosidase (lacZ) gene for histochemical identification. In one clone, C6TL beta, beta 1 protein levels were unaffected in the presence of tetracycline, but they were reduced by 60% in the absence of tetracycline because of production of antisense mRNA. C6TL beta cells were transplanted into the striatum of nude mice. After 14 days in the presence of tetracycline in the drinking water, tumors showed diffuse brain invasion, mainly along vascular basement membranes. In the absence of tetracycline, however, tumor cells were compact and generally well delineated from the surrounding brain tissue. These data, ie, blocking of brain invasion by antisense beta 1 mRNA, either because of disturbed interaction of beta 1 with brain extracellular matrix components or interference with beta 1-dependent signaling pathways, strongly suggest that beta 1 integrins are required for diffuse brain invasion of gliomas.

Topics: Animals; beta-Galactosidase; Brain; Brain Neoplasms; Cell Line; Female; Glioma; Integrin beta1; Mice; Mice, Inbred ICR; Mice, Nude; Neoplasm Invasiveness; Promoter Regions, Genetic; Rats; Recombinant Proteins; Tetracycline; Trans-Activators; Transcription, Genetic; Transfection; Transplantation, Heterologous

The successful application of phototherapy to subcutaneous tumors has suggested that a similar procedure should be developed for treating gliomas. As a result, attempts are being made to determine a set of conditions that would optimize the destruction of tumor cells while minimizing injury to surrounding brain tissue. To initiate this task, we developed a novel assay method to assess the amount of phototoxicity induced in normal brain by light exposure of mice treated with hematoporphyrin derivative (HPD). The application of this procedure demonstrated that a sufficient amount of HPD was retained in brain tissue, even 72 hours after injection, to cause severe cerebral damage in light-treated mice.

Topics: Animals; Brain; Brain Neoplasms; Glioma; Hematoporphyrins; Male; Mice; Phototherapy; Tetracycline

Topics: Brain Neoplasms; Calcium; Fluorescence; Glioma; Humans; Meningioma; Tetracycline