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OBJECTIVE Glioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid–polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth. METHODS To examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt–8 assay in mouse glioma cells. The efficacy of siRNA therapy–targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis. RESULTS In vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146–cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice. CONCLUSIONS CD146 is a potential therapeutic target, and folic acid–conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.

We report the use of small interfering RNAs (siRNAs) against ARHGEF4, CCDC88A, LAMTOR2, mTOR, NUP85, and WASF2 and folic acid (FA)-modified polyethylene glycol (PEG)-chitosan oligosaccharide lactate (COL) nanoparticles for targeting, imaging, delivery, gene silencing, and inhibition of invasiveness and metastasis in an orthotopic xenograft model. In vitro assays revealed that these siRNA-FA-PEG-COL nanoparticles were specifically inserted into pancreatic cancer cells compared to immortalized normal pancreatic epithelial cells and knocked down expression of the corresponding targets in pancreatic cancer cells. Cell motility and invasion were significantly inhibited by adding target siRNA-FA-PEG-COL nanoparticles into the culture medium. In vivo mouse experiments confirmed that when intravenously delivered, these siRNA-FA-PEG-COL nanoparticles became incorporated into human pancreatic cancer cells in mouse pancreatic tumors. Little accumulation was seen in the normal pancreas and vital organs. All target siRNA-FA-PEG-COL nanoparticles significantly inhibited retroperitoneal invasion. The siRNA-FA-PEG-COL nanoparticles against LAMTOR2, mTOR, and NUP85, which strongly inhibited retroperitoneal invasion and significantly inhibited peritoneal dissemination compared to the other nanoparticles, improved prognosis of the mice. Our results imply that siRNA-FA-PEG-COL nanoparticles against these six targets could have great potential as biodegradable drug carriers. In particular, siRNA nanoparticles against LAMTOR2, mTOR, and NUP85 may hold significant clinical promise.

Accumulating evidence indicates time-of-day variations in ischemic neuronal injury. Under ischemic conditions, Zn

Background and purpose We have demonstrated that i.c.v.-administered (±)-epibatidine, a nicotinic ACh receptor (nAChR) agonist, induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla with dihydro-β-erythroidin (an α4β2 nAChR antagonist)-sensitive brain mechanisms. Here, we examined central mechanisms for the (±)-epibatidine-induced responses, focusing on brain NOS and NO-mediated mechanisms, soluble GC (sGC) and protein S-nitrosylation (a posttranslational modification of protein cysteine thiol groups), in urethane-anaesthetized (1.0 g·kg-1 , i.p.) male Wistar rats. Experimental approach (±)-Epibatidine was i.c.v. treated after i.c.v. pretreatment with each inhibitor described below. Then, plasma catecholamines were measured electrochemically after HPLC. Immunoreactivity of S-nitrosylated cysteine (SNO-Cys) in α4 nAChR subunit (α4)-positive spinally projecting neurones in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of adrenomedullary outflow) after i.c.v. (±)-epibatidine administration was also investigated. Key results (±)-Epibatidine-induced elevation of plasma catecholamines was significantly attenuated by L-NAME (non-selective NOS inhibitor), carboxy-PTIO (NO scavenger), BYK191023 [selective inducible NOS (iNOS) inhibitor] and dithiothreitol (thiol-reducing reagent), but not by 3-bromo-7-nitroindazole (selective neuronal NOS inhibitor) or ODQ (sGC inhibitor). (±)-Epibatidine increased the number of spinally projecting PVN neurones with α4- and SNO-Cys-immunoreactivities, and this increment was reduced by BYK191023. Conclusions and implications Stimulation of brain nAChRs can induce elevation of plasma catecholamines through brain iNOS-derived NO-mediated protein S-nitrosylation in rats. Therefore, brain nAChRs (at least α4β2 subtype) and NO might be useful targets for alleviation of catecholamines overflow induced by smoking.

Microglia, the resident immune cells of the central nervous system, can display a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. Arginase (Arg)-1 expressed in interleukin-4 (IL-4)-induced M2 microglia reduces nitric oxide (NO) production by competing with inducible NO synthase for l-arginine, which contributes to the attenuation of brain inflammation. Although previous studies have indicated that brain zinc promotes M1 activation, the effect of zinc on M2 microglial activation remains to be determined. In the present study, murine primary microglia treated with 10 ng mL-1 IL-4 exhibited increased Arg-1 mRNA expression and levels of intracellular free zinc. Chelation of this increased intracellular free zinc by the cell permeable zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) aggravated the IL-4-induced mRNA expression and enzymatic activity of Arg-1. However, the cell impermeable zinc chelator CaEDTA had no effect on Arg-1 expression or cytosolic levels of free zinc in IL-4-induced M2-polarized microglia. Furthermore, treatment with IL-4 resulted in upregulation of phagocytic activity in microglia, while administration of TPEN abolished IL-4-induced phagocytic activity. Moreover, this effect was reversed vial-arginine supplementation. These findings suggest that IL-4 induces an increase in intracellular free zinc in microglia, which may act as a negative regulator of IL-4-induced Arg-1 expression, and that such negative regulation is essential for microglial phagocytic activity.

Background Glial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation. Purpose Here, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity. Methods Engulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl2-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox. Results Although treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl2 was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl2. Conclusion These findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.

Accumulating evidence indicates time-of-day variations in ischemic neuronal injury. Under ischemic conditions, Zn2+ is massively released from hippocampal glutamatergic neurons, and intracellular Zn2+ accumulation results in neuron death. Notably, excitatory amino acid carrier 1 (EAAC1), known as a cysteine transporter, is involved in Zn2+ homeostasis, and its expressions exhibit a diurnal fluctuation. This study aimed to investigate whether time of day of an ischemic insult affects Zn2+ accumulation and neuronal injury and determine whether altered Zn2+ accumulation is modulated by EAAC1 diurnal fluctuation in the hippocampus in a mouse model of ischemic stroke. Mice subjected to transient global ischemia for 40 min at Zeitgeber time 18 (ZT18) (23:00) exhibited reduced Zn2+ accumulation and neuronal death in the hilar region of the hippocampus compared to those at ZT4 (09:00). The EAAC1 protein expression in the hippocampus was increased at ZT18 relative to ZT4. Intracerebroventricular injection of a non-selective excitatory amino acid transporter inhibitor, DL-threo-β-benzyloxyaspartate, or a selective EAAC1 inhibitor, L-aspartic acid β-hydroxamate, increased ischemia-induced Zn2+ accumulation and neuronal death in the hilus at ZT18. These findings suggest that ischemia-induced Zn2+ accumulation displays circadian fluctuations through diurnal variations in EAAC1 expressions and affects susceptibility to ischemic neuronal injury in the hippocampal hilar region.

INTRODUCTION Astroblastoma is a rare, mostly supratentorial glial tumor, occurring predominantly in children and young adults. However, treatment strategies have not yet been established for this rare disease. CASE PRESENTATION A 6-year-old male presented with headache and nausea. CT and MR imaging revealed a left frontal mass lesion with slight edema and macrocalcifications. Gross tumor resection was performed. Histological examination found neoplastic cells with astroblastic characteristics, and a striking perivascular array of pseudorosettes. The final diagnosis was high-grade astroblastoma. MR imaging 13 months after surgery suggested local recurrence and enlargement was found 3 months later. Stereotactic radiotherapy (SRT) was performed. MR imaging after SRT showed enhanced cyst formation around the tumor bed, suggesting tumor recurrence. However, 11C-methionine PET revealed radiation necrosis. The last follow-up MR imaging 15 months after SRT showed no further recurrence. CONCLUSION Astroblastoma is rare, so no optimal management is known. SRT may be effective to treat recurrent astroblastomas. 11C-methionine PET/CT is useful for the differentiation from radiation necrosis.

CD146 is highly expressed in various malignant tumors and contributes to their malignancy phenotype, which involves metastatic and tumorigenic activity. However, studies on the expression and function of CD146 in brain tumors are limited. We over-expressed or knocked-down CD146 in both conventionally cultured glioma cells and tumor spheres (TS). The distribution of glioma cells and their stem cells in different cell cycle phases was analyzed by flow cytometry using the stem cell marker CD133 and the glial precursor marker A2B5. CD146 expression was immunohistochemically examined in glioma tissues. The majority of glioma stem cells (GSCs) expressing CD133 were also CD146-positive. CD146 knockdown in GSCs significantly compromised cell growth. Cell cycle analysis revealed that most of the CD146 and CD133 double-positive cells were in the G2/M phase. Ectopic expression of CD146 in parental glioma cells resulted in cell cycle arrest of most differentiated cells in G0/G1 phase. In contrast, ectopic expression of CD146 in GSCs resulted in an increase in the number of CD133-positive cells in the G2/M phase. Furthermore, CD146 knockdown reduced the number of CD133-positive cells in the G2/M phase, which was consistent with effects of cell growth inhibition. Immunohistochemical analysis revealed that CD146 expression was significantly upregulated in World Health Organization (WHO) Grade III and IV glioma and positively correlated with CD133 expression. CD146 is mainly expressed in dividing GSCs and may be a potential target for eradicating glioma stem cells.

OBJECT Wilms’ tumor 1 (WT1) peptide vaccination is considered a potentially effective therapy against malignant glioma. We conducted a Phase I/II study to investigate the safety and feasibility of novel WT1 peptide (W10) vaccination therapy for patients with newly diagnosed malignant glioma. METHODS WT1 vaccination therapy was performed for patients with malignant glioma who have undergone concurrent radiotherapy and temozolomide therapy. A mixture of WT1 peptide with inactivated pertussis whole cell vaccine was injected intradermally once a week for at least 12 weeks. RESULTS Twenty-seven patients (12 men, 15 women; median 65 years) with the following tumors were enrolled: WHO grade IV (15), WHO grade III (12). PFS and OS of glioblastoma cases were 12.7 months 21.9 months, respectively. PFS of the MGMT unmethylated group was shorter than the methylated group. Interestingly enough, overall survival in the MGMT unmethylated group was not significantly different from the methylated group. Analysis of recurrent cases after immunotherapy showed decreased expression of WT1 antigen and increased Treg. They were suggested as a cause of treatment resistance. No serious adverse events were observed except for Grade 1 erythema at the injection sites. CONCLUSIONS This study of a novel WT1 vaccination therapy demonstrated safety and feasibility in the management of newly diagnosed malignant gliomas.

BACKGROUND AND PURPOSE: We have demonstrated that i.c.v.‐administered (±)‐epibatidine, a nicotinic ACh receptor (nAChR) agonist, induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla with dihydro‐β‐erythroidin (an α4β2 nAChR antagonist)‐sensitive brain mechanisms. Here, we examined central mechanisms for the (±)‐epibatidine‐induced responses, focusing on brain NOS and NO‐mediated mechanisms, soluble GC (sGC) and protein S‐nitrosylation (a posttranslational modification of protein cysteine thiol groups), in urethane‐anaesthetized (1.0 g·kg(−1), i.p.) male Wistar rats. EXPERIMENTAL APPROACH: (±)‐Epibatidine was i.c.v. treated after i.c.v. pretreatment with each inhibitor described below. Then, plasma catecholamines were measured electrochemically after HPLC. Immunoreactivity of S‐nitrosylated cysteine (SNO‐Cys) in α4 nAChR subunit (α4)‐positive spinally projecting neurones in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of adrenomedullary outflow) after i.c.v. (±)‐epibatidine administration was also investigated. KEY RESULTS: (±)‐Epibatidine‐induced elevation of plasma catecholamines was significantly attenuated by L‐NAME (non‐selective NOS inhibitor), carboxy‐PTIO (NO scavenger), BYK191023 [selective inducible NOS (iNOS) inhibitor] and dithiothreitol (thiol‐reducing reagent), but not by 3‐bromo‐7‐nitroindazole (selective neuronal NOS inhibitor) or ODQ (sGC inhibitor). (±)‐Epibatidine increased the number of spinally projecting PVN neurones with α4‐ and SNO‐Cys‐immunoreactivities, and this increment was reduced by BYK191023. CONCLUSIONS AND IMPLICATIONS: Stimulation of brain nAChRs can induce elevation of plasma catecholamines through brain iNOS‐derived NO‐mediated protein S‐nitrosylation in rats. Therefore, brain nAChRs (at least α4β2 subtype) and NO might be useful targets for alleviation of catecholamines overflow induced by smoking.

Previously, we reported that central administration of bombesin, a stress-related peptide, elevated plasma levels of catecholamines (noradrenaline and adrenaline) in the rat. The sympatho-adrenomedullary system, which is an important component of stress responses, can be regulated by the central opioid system. In the present study, therefore, we examined the roles of brain opioid receptor subtypes (µ, δ, and κ) and nociceptin receptors, originally identified as opioid-like orphan receptors, in the bombesin-induced activation of central sympatho-adrenomedullary outflow using anesthetized male Wistar rats. Intracerebroventricularly (i.c.v.) administered bombesin-(1 nmol/animal) induced elevation of plasma catecholamines was significantly potentiated by pretreatment with naloxone (300 and 1000 µg/animal, i.c.v.), a non-selective antagonist for µ-, δ-, and κ-opioid receptors. Pretreatment with cyprodime (100 µg/animal, i.c.v.), a selective antagonist for µ-opioid receptors, also potentiated the bombesin-induced responses. In contrast, pretreatment with naltrindole (100 µg/animal, i.c.v.) or nor-binaltorphimine (100 µg/animal, i.c.v.), a selective antagonist for δ- or κ-opioid receptors, significantly reduced the elevation of bombesin-induced catecholamines. In addition, pretreatment with JTC-801 (30 and 100 µg/animal, i.c.v.) or J-113397 (100 µg/animal, i.c.v.), which are selective antagonists for nociceptin receptors, also reduced the bombesin-induced responses. These results suggest that brain µ-opioid receptors play a suppressive role and that brain δ-, κ-opioid, and nociceptin receptors play a facilitative role in the bombesin-induced elevation of plasma catecholamines in the rat. Thus, in the brain, these receptors could play differential roles in regulating the activation of central sympatho-adrenomedullary outflow.

Cancer stem cells (CSCs) are the sole population possessing high self-renewal activity in tumors, with their existence affecting tumor recurrence. However, the invasive activity of CSCs has yet to be fully understood. In this article, we established a tumor sphere culture of RSV-M mouse glioma cells (RSV-M-TS) and evaluated their migration and invasion activities. Histological analysis of a tumor formed by cranial injection of the RSV-M-TS cells showed highly invasive properties and similarities with human malignant glioma tissues. When the migration activity of both RSV-M and RSV-M-TS cells were compared by intracranial injection, rapid migration of RSV-M-TS cells was observed. To confirm the invasive capabilities of RSV-M-TS cells, a three-dimensional collagen invasion assay was performed in vitro using RSV-M, RSV-M-TS, and RSV-M-TS cells cultured with medium containing serum. RSV-M and RSV-M-TS cultured with medium containing serum for 8 days indicated low migration activity, while moderate invasion activity was observed in RSV-M-TS cells. This activity was further enhanced by incubation with medium containing serum overnight. To identify the genes involved in this invasion activity, we performed quantitative polymerase chain reaction (PCR) array analysis of RSV-M and RSV-M-TS cells. Of 84 cancer metastasis-related genes, up-regulation was observed in 24 genes, while 4 genes appeared to be down-regulated in RSV-M-TS cells. These results suggest that the enhanced invasive activity of glioma sphere cells correlates with a number of tumor metastasis-related genes and plays a role in the dissemination and invasion of glioma cells.

Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30–60 μM ZnCl2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia–reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory.

Brain concussions are a serious public concern and are associated with neuropsychiatric disorders, such as depression. Patients with concussion who suffer from depression often experience distress. Nevertheless, few pre-clinical studies have examined concussion-induced depression, and there is little information regarding its pharmacological management. Edaravone, a free radical scavenger, can exert neuroprotective effects in several animal models of neurological disorders. However, the effectiveness of edaravone in animal models of concussion-induced depression remains unclear. In this study, we examined whether edaravone could prevent concussion-induced depression. Mice were subjected to a weight-drop injury and intravenously administered edaravone (3.0 mg/kg) or vehicle immediately after impact. Serial magnetic resonance imaging showed no abnormalities of the cerebrum on diffusion T1- and T2-weighted images. We found that edaravone suppressed concussion-induced depressive-like behavior in the forced swim test, which was accompanied by inhibition of increased hippocampal and cortical oxidative stress (OS) and suppression of 5-lipoxygenase (5-LOX) translocation to the nuclear envelope in hippocampal astrocytes. Hippocampal OS in concussed mice was also prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin, and administration of BWB70C, a 5-LOX inhibitor, immediately and 24 h after injury prevented depressive-like behaviors in concussed mice. Further, antidepressant effects of edaravone were observed in mice receiving 1.0 or 3.0 mg/kg of edaravone immediately after impact, but not at a lower dose of 0.1 mg/kg. This antidepressant effect persisted up to 1 h after impact, whereas edaravone treatment at 3 h after impact had no effect on concussion-induced depressive-like behavior. These results suggest that edaravone protects against concussion-induced depression, and this protection is mediated by suppression of OS and 5-LOX translocation.

For effective ovarian cancer gene therapy, systemic administrated tumor-targeting siRNA/folic acid-poly(ethylene glycol)-chitosan oligosaccharide lactate (FA-PEG-COL) nanoparticles is vital for delivery to cancer site(s). siRNA/FA-PEG-COL nanoparticles were prepared by ionic gelation for effective FA receptor-expressing ovarian cancer cells transfection and in vivo accumulation. The chemical structure of FA-PEG-COL conjugate was characterized by MALDI-TOF-MS, FT-IR and (1)H NMR. The average size of siRNA/FA-PEG-COL nanoparticles was approximately 200 nm, and the surface charge was +8.4 mV compared to +30.5 mV with siRNA/COL nanoparticles. FA-PEG-COL nanoparticles demonstrated superior compatibility with erythrocytes in terms of degree of aggregation and haemolytic activity and also effects on cell viability was lower when compared with COL nanoparticles. FA grafting significantly facilitated the uptake of nanoparticles via receptor mediated endocytosis as demonstrated by flow cytometry. The in vitro transfection and gene knockdown efficiency of HIF-1α were superior to COL nanoparticles (76-62%, respectively) and was comparable to Lipofectamine 2000 (79%) as demonstrated by RT-qPCR and Western blot. Gene knockdown at the molecular level translated into effective inhibition of proliferation in vitro. Accumulation efficiency of FA-PEG-COL nanoparticles was investigated in BALB/c mice bearing OVK18 #2 tumor xenograft using in vivo imaging. The active targeting FA-PEG-COL nanoparticles showed significantly greater accumulation than the passive targeting COL nanoparticles. Based on the results obtained, siRNA/FA-PEG-COL nanoparticles show much potential for effective ovarian cancer treatment via gene therapy.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to improve the prognosis of GBM, however, with limited success. Many gene therapy strategies for GBM have been developed and a few have progressed to clinical trials. Retroviral vectors have superior features for gene therapy in brain cancers, including tumor specificity, immunogenicity, and longer half-life. Early gene therapy trials in GBM patients based on transplantation of retrovirus-producing cells into the brain failed to prove efficacious. Adenoviral vectors, which can be prepared as high-titer virus solutions and undergo efficient transduction in tumor cells, failed in clinical trials, likely due to immunogenicity and instability of gene expression. Alternative therapeutics such as oncolytic viruses that specifically target and destroy cancer cells are currently under investigation. In addition to novel vectors, retroviral vectors are still attractive candidates for use in gene therapy against brain tumors. Since yields of properly-packaged viral particles from virus-producing cells have been very limited so far, gene therapy by direct injection of hightiter retroviral vectors into the patients’ brains was not possible. To overcome these disadvantages, a packaging cell line that yields high-titer retroviral solutions was established by our group, enabling the direct injection of massive retroviral vector stocks directly into the brain. Mouse glioma models were effectively cured with a combination of a suicide gene/ prodrug system and a highly-concentrated retrovirus solution. Preclinical assessments, including that of replication-competent retroviruses and tumorigenicity of the combination method, have confirmed the safety of the highly-concentrated retrovirus solution. Addi tional studies are needed to address the clinical utility of such combination gene therapies. Taken together, these data suggest that retroviral vectors are still good candidates for development in gene therapy applications.

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down-regulating angiogenesis via a Toll-like receptor 4 signal. Murine RSV-M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV-M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)-17 in both C3H/HeN and C3H/HeJ tumor-bearing mice. Treatment with E. coli LPS induced much greater IL-17 production in tumor-bearing C3H/HeN mice than in tumor-bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re-transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti-cluster of differentiation (CD)8, anti-CD4, anti-CD8 antibodies, and anti-asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti-interferon-γ antibodies had no effect on glioma cell growth, anti-IL-17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS-treated mice than in those from saline- or E. coli LPS-treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down-regulating angiogenesis, and that this down-regulation is mediated in part by regulating IL-17 production.

Suicide gene therapy has been shown to be effective in inducing tumor regression. In this study, a human brain tumor-specific promoter was identified and used to develop transcriptionally targeted gene therapy. We searched for genes with brain tumor-specific expression. By in silico and reverse-transcription polymerase chain reaction screening, MAGE-A3 and SSX4 were found to be expressed in a tumor-specific manner. SSX4 gene promoter activity was high in human brain tumor cells but not in normal human astrocyte cells, whereas the MAGE-A3 promoter showed activity in both tumor and normal cells. A retrovirus vector carrying a suicide gene, the herpes simplex virus thymidine kinase gene controlled by the SSX4 promoter, was constructed to evaluate the efficacy of the promoter in tumor-specific gene therapy. Glioma and human telomerase catalytic subunit-immortalized fibroblast BJ-5ta cell lines transduced with retrovirus vectors were assayed for killing activity by ganciclovir. Glioma cell lines were effectively killed by ganciclovir in a concentration-dependent manner, whereas BJ-5ta cells were not. By contrast, MAGE-A3 promoter failed to induce cytotoxicity in a brain tumor-specific manner. In addition, mouse glioma RSV-M cells transduced with retrovirus vector also showed suppressed tumor formation activity in syngeneic mice in response to ganciclovir administration. Therefore, the SSX4 promoter is a candidate for brain tumor-specific gene therapy and supports the efficacy and safety of suicide gene therapy for malignant brain tumors.

Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance, and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression of cancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly and frequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels in the promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNA methylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic difference between cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at various levels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiation status, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findings suggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in glioma patients.

We previously reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (1, 5 or 10 nmol/animal), a nicotinic acetylcholine receptor agonist, dose-dependently induced secretion of noradrenaline and adrenaline (catecholamines) from the rat adrenal medulla by brain diacylglycerol lipase- (DGL), monoacylglycerol lipase- (MGL) and cyclooxygenase-mediated mechanisms. Diacylglycerol is hydrolyzed by DGL into 2-arachidonoylglycerol (2-AG), which is further hydrolyzed by MGL to arachidonic acid (AA), a cyclooxygenase substrate. These findings suggest that brain 2-AG-derived AA is involved in the (±)-epibatidine-induced response. This AA precursor 2-AG is also a major brain endocannabinoid, which inhibits synaptic transmission through presynaptic cannabinoid CB1 receptors. Released 2-AG into the synaptic cleft is rapidly inactivated by cellular uptake. Here, we examined a role of brain 2-AG as an endocannabinoid in the (±)-epibatidine-induced activation of central adrenomedullary outflow using anesthetized male Wistar rats. In central presence of AM251 (CB1 antagonist) (90 and 180 nmol/animal, i.c.v.), (±)-epibatidine elevated plasma catecholamines even at an ineffective dose (1 nmol/animal, i.c.v.). Central pretreatment with ACEA (CB1 agonist) (0.7 and 1.4 μmol/animal, i.c.v.), 2-AG ether (stable 2-AG analog for MGL) (0.5 and 1.0 μmol/animal, i.c.v.) or AM404 (endocannabinoid uptake inhibitor) (80 and 250 nmol/animal, i.c.v.) significantly reduced an effective dose of (±)-epibatidine- (5 nmol/animal, i.c.v.) induced elevation of plasma catecholamines, and AM251 (90 and 180 nmol/animal, i.c.v.) centrally abolished the reduction induced by 2-AG ether (1.0 μmol/animal, i.c.v.) or AM404 (250 nmol/animal, i.c.v.). Immunohistochemical studies demonstrated that (±)-epibatidine (10 nmol/animal, i.c.v.) activated DGLα-positive spinally projecting neurons in the hypothalamic paraventricular nucleus, a control center of central adrenomedullary system. These results suggest a possibility that a brain endocannabinoid, probably 2-AG, plays an inhibitory role in (±)-epibatidine-induced activation of central adrenomedullary outflow through brain CB1 receptors in the rat.

Introduction: We identified CCDC88A messenger RNA (mRNA) that accumulates in lamellipodia by binding to an RNA-binding protein IGF2BP3 with a next generation sequencer. The objectives of this study are as follows: (1) To identify CCDC88A specific small interfering RNAs (siRNAs) as RNA interference agents that have an inhibitory effect on the invasion and metastasis of pancreatic cancer cells in a mouse model of the invasion and metastasis of pancreatic cancer; and (2) To develop a nucleic-acid delivery system to efficiently transport the siRNAs to PDAC tissue. Methods: After intravenous injection of CCDC88A-specific siRNAs supplemented with folic acids that can bind to the folic acid receptor—which are highly expressed on pancreatic cancer cells—and polyethylene glycol-chitosan oligosaccharide nanoparticles to a mouse model of invasion and metastasis in which a human pancreatic cell line was transplanted to induce tumor formation in the mouse pancreas, accumulation of the siRNAs in the pancreatic cancer tissue was determined by using an in vivo imaging system. After CCDC88A-specific siRNA accumulation in the pancreatic cancer tissue was identified, invasion to the retroperitoneum as well as metastasis to the lung or liver from the human primary pancreatic tumor was compared between the mice in which CCDC88A-specific siRNA accumulation was identified and those receiving a scrambled control siRNA. Results: CCDC88A-specific siRNAs could be delivered to the pancreatic cancer cells of cancer-bearing nude mice by supplementation with folic acids and chitosan oligosaccharide nanoparticles. Supplementation with folic acids and chitosan oligosaccharide nanoparticles caused no hemolysis and had no effect on liver, kidney, or pancreatic function. Invasion to the retroperitoneum and metastasis to the lung or liver from the primary pancreatic cancer were suppressed in cancer-bearing nude mice by administration of those siRNAs, which target CCDC88A mRNA accumulated in lamellipodia of pancreatic cancer cells. Conclusions: This study suggests the potential to develop a siRNA drug to inhibit the invasion and metastasis of pancreatic cancer. Citation Format: Keisuke Taniuchi, Toshio Yawata, Tetsuya Ueba, Toshiji Saibara. Efficient delivery of siRNA targeted CCDC88A mRNA to pancreatic cancer tumor inhibits the invasiveness and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5131. doi:10.1158/1538-7445.AM2017-5131

Angiotensin II (AngII) plays important roles in the regulation of cardiovascular function. Both peripheral and central actions of AngII are involved in this regulation, but mechanisms of the latter actions as a neurotransmitter/neuromodulator within the brain are still unclear. Here we show that (1) intracerebroventricularly (i.c.v.) administered AngII in urethane-anesthetized male rats elevates plasma adrenaline derived from the adrenal medulla but not noradrenaline with valsartan- (AT1 receptor blocker) sensitive brain mechanisms, (2) peripheral AT1 receptors are not involved in the AngII-induced elevation of plasma adrenaline, although AngII induces both noradrenaline and adrenaline secretion from bovine adrenal medulla cells, and (3) i.c.v. administered AngII elevates blood pressure but not heart rate with the valsartan-sensitive mechanisms. From these results, i.c.v. administered AngII acts on brain AT1 receptors, thereby inducing the secretion of adrenaline and pressor responses. We propose that the central angiotensinergic system can activate central adrenomedullary outflow and modulate blood pressure.

Background The association between human cytomegalovirus (HCMV) and glioblastoma multiforme (GBM) is becoming a new concept. However, information on the geographic variability of HCMV prevalence in GBM remains scarce. Moreover, the potential roles of various viruses, such as polyomaviruses and oncogenic viruses, in gliomagenesis remain unclear. Our aim was to investigate the prevalence of HCMV in GBM among Japanese patients. Furthermore, this was the first study that evaluated infection with four new human polyomaviruses in GBMs. This study also provided the first data on the detection of human papillomavirus (HPV) in GBM in the Eastern world. Methods We measured the number of various viral genomes in GBM samples from 39 Japanese patients using real-time quantitative PCR. The tested viruses included HCMV, Merkel cell polyomavirus, human polyomavirus (HPyV) 6, HPyV7, HPyV9, Epstein–Barr virus, human herpesvirus 8, and HPV. Our quantitative PCR analysis led to the detection of eight copies of the HCMV DNA mixed with DNA extracted from 104 HCMV-negative cells. The presence of HCMV and HPV genomes was also assessed by nested PCR. Immunohistochemical study was also carried out to detect HPV-derived protein in GBM tissues. Results The viral DNAs were not detectable, with the exception of HPV, which was present in eight out of 39 (21%) GBMs. All HPV-positive cases harbored high-risk-type HPV (HPV16 and HPV18). Moreover, the HPV major capsid protein was detected in GBM tumor cells. Conclusions In contrast with previous reports from Caucasian patients, we did not obtain direct evidence in support of the association between HCMV and GBM. However, high-risk-type HPV infection may play a potential etiological role in gliomagenesis in a subset of patients. These findings should prompt further worldwide epidemiological studies aimed at defining the pathogenicity of virus-associated GBM.

Normal human somatic cells stop dividing after a limited number of cell divisions through the process termed "cellular senescence," which may function as a tumor-suppressive mechanism. Although the regulation of telomerase activity and telomere length plays an important role in cellular senescence and immortalization in human cells, recent findings from the telomerase introduction experiment, chromosome transfer experiment and studies on the inducers of premature senescence suggest that there are multiple mechanisms to induce cellular senescence, some of which are independent of telomerase and telomere regulation. Inactivation of the function of cell cycle regulatory pl6INK4A/Rb pathway is a key event in immortalization. In rodents, which have long telomeres and telomerase activity, telomere shortening is not correlated with cellular senescence. These models may be useful to elucidate telomere-independent pathways in human cells. Studies in this field will clarify the barriers to human cell immortalizatio...

We recently reported that intracerebroventricularly administered 2-arachidonoylglycerol elevated plasma noradrenaline and adrenaline by brain monoacylglycerol lipase- (MGL) and cyclooxygenase-mediated mechanisms in the rat. These results suggest that 2-arachidonoylglycerol is hydrolyzed by MGL to free arachidonic acid, which is further metabolized to prostaglandins (PGs) by cyclooxygenase in the brain, thereby elevating plasma noradrenaline and adrenaline. On the other hand, 2-arachidonoylglycerol can be also metabolized by cyclooxygenase to PG glycerol esters (PG-Gs), which seems to be hydrolyzed by MGL to free PGs. Here, we examined the involvement of brain PG-Gs in the elevation of plasma noradrenaline and adrenaline regarding PGE2-G and prostanoid EP receptors using anesthetized male Wistar rats. Intracerebroventricularly administered PGE2-G (1.5 and 3 nmol/animal) dose-dependently elevated plasma noradrenaline but not adrenaline. PGE2-G also elevated systolic, mean and diastolic blood pressure and heart rate. The PGE2-G-induced elevation of plasma noradrenaline was attenuated by JZL184 (MGL inhibitor). Intracerebroventricularly administered PGE2 (0.3 and 1.5 nmol/animal) and sulprostone (0.1 and 0.3 nmol/animal) (EP1/EP3 agonist) also elevated plasma noradrenaline but not adrenaline in a dose-dependent manner. The sulprostone-induced elevation was attenuated by L-798,106 (EP3 antagonist), but not by SC-51322 (EP1 antagonist). L-798,106 also attenuated the PGE2-G- and PGE2-induced elevation of plasma noradrenaline, while PF-04418948 (EP2 antagonist) and L-161,982 (EP4 antagonist) had no effect on the PGE2-G-induced response. These results suggest a possibility that brain PGE2-G produced from 2-arachidonoylglycerol can be hydrolyzed to free PGE2, thereby activating central sympathetic outflow by brain prostanoid EP3 receptor-mediated mechanisms in the rat.

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down-regulating angiogenesis via a Toll-like receptor 4 signal. Murine RSV-M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV-M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)-17 in both C3H/HeN and C3H/HeJ tumor-bearing mice. Treatment with E. coli LPS induced much greater IL-17 production in tumor-bearing C3H/HeN mice than in tumor-bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re-transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti-cluster of differentiation (CD)8, anti-CD4, anti-CD8 antibodies, and anti-asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti-interferon-γ antibodies had no effect on glioma cell growth, anti-IL-17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS-treated mice than in those from saline- or E. coli LPS-treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down-regulating angiogenesis, and that this down-regulation is mediated in part by regulating IL-17 production.

Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance, and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression of cancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly and frequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels in the promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNA methylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic difference between cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at various levels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiation status, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findings suggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in glioma patients.

We established a cancer stem (CS) cell line, U87CS, by means of spheroid culture of U87MG cells derived from glioblastoma (GBM) in neuronal stem cell medium. U87CS cells presented positive immunohistochemical staining for multidrug resistance (MDR)1 and CD133, a marker for a subset of leukemia and GBM CS cells. The gene expression of MDR1 and CD133 on U87CS cells increased by an average of 8.51 and 47.18 times, respectively, compared to the levels on U87MG cells by real-time quantitative RT-PCR. U87CS cells possessed stronger drug-resistance to conventional anti-cancer drugs, such as doxorubicin (Dox), etoposide (VP-16), carboplastin, and BCNU than U87MG cells. Double immunofluoresence staining showed co-expression of MDR1 and CD133 on U87CS cells transplanted into nude mice brains. In addition, we identified the crossreactivity of CD133 and MDR1 in a surgical specimen of GBM. Our results suggest that CS cells may be resistant to current chemotherapy and represent a novel target for GBM therapeutics.

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.

To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to theneo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH-(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.

Cancer stem cell hypothesis suggests that small populations of stem-like cells exist in the tumor mass and harbor drug resistance and low radio-sensitivity, and thus resulting in tumor recurrence after various treatments. In this study, we investigated the relationship between cell differentiation and drug resistant gene expression in glioblastoma. Glioma stem-like cells freshly isolated from surgical specimens were enriched in tumor sphere (TS) culture and were more resistant to anti-cancer drugs compared with their differentiated cells in cell viability assay. These cells were also tested for phosphorylation of histone H2AX at Ser139, a biomarker of DNA lesions including DNA double-strand and single-strand breaks. Differentiated cells were much more susceptible to anti-cancer drug-induced DNA damage, compared with TS cells. The expression of drug resistance genes, MDR-1, MRP-1 and MRP-2 in TS cells was more than those in their differentiated cells by RT-PCR analysis. However, there was no correlation between drug resistance genes and stem cell marker CD133 expression. It is possibly caused by another cell population expressing drug resistance genes, therefore, we sought to define the existence of progenitor cells in glioma by detection of glial progenitor marker, A2B5 using flow cytometric analysis. CD133 and A2B5 positive cells were more abundant in TS cells and were decreased during induction of differentiation. The expression of MDR-1 was frequently observed in CD133+/A2B5−, CD133+/A2B5+ and CD133−/A2B5+ cells. Reduction of MDR-1 expression concomitantly occurred with the differentiation of these immature glioma cells. Thus, it is revealed that CD133 negative cells expressing MDR-1 are A2B5-positive glioma cells. These results suggest that drug resistance genes are expressed not only in tumor stem cells but also in progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5362. doi:1538-7445.AM2012-5362

Glioblastomas are characterized by aggressive invasion into normal brain tissues, thus limiting complete removal by surgical resection resulting in recurrence. Our identified molecule involved in glioma invasion is important for developing targeted effective therapies in the malignant glioma. Cancer stem cells are capable for self-renewing and are thought to be responsible for tumor metastasis and dissemination. However, the invasive activities of cancer stem cells have not been fully understood. To investigate a role of glioma stem cells in the tumor invasion, tumor sphere culture was established from mouse glioma RSV-M cells. The high tumorigeneity and aggressive migration of RSV-M tumor sphere were observed in intracranial transplantation. Quantitative RT-PCR analysis showed the enhanced expression of metastasis-related genes in RSV-M tumor sphere. Of these, MUC18/MCAM also highly expressed in tumor sphere derived from human glioma cell lines. Importantly, CD133-positive fraction of glioma sphere culture isolated from glioblastoma patients contained a cell population expressing MUC18 but CD133-negative fraction not. To examine a function of MUC18 in glioma cells, the expression or knockdown construct for the gene were introduced into human glioma cells. Over-expression of MUC18 in the glioma cells increased their invasive capacity and clonogenicity in comparison to the parent or knockdown cells. In addition, expression of some metastasis-related genes was up-regulated in MUC18-over-expressing cells. These results suggest that enhanced MUC18 expression of glioma stem cells may contribute to the aggressive invasion of this tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3402. doi:10.1158/1538-7445.AM2011-3402

Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We first searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes by in silico and RT-PCR screening. MAGEA3 and SSX4 were identified as tumor-specific genes. The promoter activity was measured in glioma, telomerase-immortalized fibroblast and NHA cells. The SSX4 promoter but not MAGEA3 showed the tumor-specific activity. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomerase-immortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into S.C. of syngeneic mouse. The administration of GCV suppressed the tumor growth completely. These results suggest that trans-acting factors may play a role for tumor-specific activity of SSX4 promoter while the expression of most CTA genes is regulated by epigenetic factors and the retrovirus vector expressing HSVtk under control of SSX4 promoter is a promising therapeutic reagent for malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 587.