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CCRT (concomitant chemotherapy and radiation therapy) is often used for glioblastoma multiforme (GBM) treatment after surgical therapy, however, patients treated with CCRT undergo poor prognosis due to development of treatment resistant recurrence. Many studies have been performed to overcome these problems and to discover genes influencing treatment resistance. To discover potential genes inducing CCRT resistance in GBM, we used whole genome screening by infecting shRNA pool in patient-derived cell. The cells infected ~8,000 shRNAs were implanted in mouse brain and treated RT/TMZ as in CCRT treated patients. We found DDX6 as the candidate gene for treatment resistance after screening and establishing DDX6 knock down cells for functional validation. Using these cells, we confirmed tumor associated ability of DDX6 in vitro and in vivo. Although proliferation improvement was not found, decreased DDX6 influenced upregulated clonogenic ability and resistant response against radiation treatment in vivo and in vitro. Taken together, we suggest that DDX6 discovered by using whole genome screening was responsible for radio- and chemoresistance in GBM.

Tumor-tropic properties of neural stem cells (NSCs) provide a novel approach with which to deliver targeting therapeutic genes to brain tumors. Previously, we developed a therapeutic strategy against metastatic brain tumors using a human NSC line (F3) expressing cytosine deaminase (F3.CD). F3.CD converts systemically administered 5-fluorocytosine (5-FC), a blood-brain barrier permeable nontoxic prodrug, into the anticancer agent 5-fluorouracil (5-FU). In this study, we potentiated a therapeutic strategy of treatment with nucleosides in order to chemically facilitate the endogenous conversion of 5-FU to its toxic metabolite 5-FU ribonucleoside (5-FUR). In vitro, 5-FUR showed superior cytotoxic activity against MDA-MB-435 cancer cells when compared to 5-FU. Although adenosine had little cytotoxic activity, the addition of adenosine significantly potentiated the in vitro cytotoxicity of 5-FU. When MDA-MB‑435 cells were co-cultured with F3.CD cells, F3.CD cells and 5-FC inhibited the growth of MDA-MB-435 cells more significantly in the presence of adenosine. Facilitated 5-FUR production by F3.CD was confirmed by an HPLC analysis of the conditioned media derived from F3.CD cells treated with 5-FC and adenosine. In vivo systemic adenosine treatment also significantly potentiated the therapeutic effects of F3.CD cells and 5-FC in an MDA-MB-435 metastatic brain tumor model. Simple adenosine addition improved the antitumor activity of the NSCs carrying the therapeutic gene. Our results demonstrated an increased therapeutic potential, and thereby, clinical applicability of NSC-based gene therapy.

Frequent early development of systemic metastasis leads to unfavourable clinical prognosis of non-small cell lung cancer (NSCLC). Although brain metastasis (BM) contributes significantly to morbidity and mortality of NSCLC, relevant driver mechanisms are largely unknown. To elucidate genetic alterations associated with early BM of NSCLC, we retrospectively collected 18 NSCLC cases with BM [12 adenocarcinomas (ADC) and 6 squamous cell carcinomas (SQCC)] whose surgical tissues of both primary and brain metastatic tumors were preserved as formaldehyde-fixed and paraffin-embedded (FFPE) pathological samples. When chromosomal copy number alterations (CNA) of those FFPE samples were analysed by the Molecular Inversion Probe (MIP) technology, the most frequent CNAs detected in primary lung ADCs were gains of 3q, 5p, 5q, 6p, 8q, 9p, 11p, 15q, 17q and losses of 10q and 22q whereas primary lung SQCCs revealed gains in 4q and 12q and loss in 9q. In particular, when comparative MIP was performed in primary 12 ADCs depending on the pattern of BM to uncover predetermining signatures that can predict the risk of BM, selectively amplified regions of primary lung ADCs (5q35, 10q23 and 17q23-24) were identified as significantly associated with the development of early BM within 3 months after first diagnosis of primary tumors. Those regions harbour several candidate genes including NeurL1B, ACTA2, FAS and ICAM2. Although more validation is needed, the genetic signatures elucidated in this study help to identify useful molecular markers defining an NSCLC patient subgroup at risk of early BM, guiding therapeutic decisions.

A set of proteins reflecting the prognosis of patients have clinical significance since they could be utilized as predictive biomarkers and/or potential therapeutic targets. With the aim of finding novel diagnostic and prognostic markers for glioblastoma (GBM), a tissue microarray (TMA) library consisting of 62 GBMs and 28 GBM-associated normal spots was constructed. Immunohistochemistry against 78 GBM-associated proteins was performed. Expression levels of each protein for each patient were analyzed using an image analysis program and converted to H-score [summation of the intensity grade of staining (0-3) multiplied by the percentage of positive cells corresponding to each grade]. Based on H-score and hierarchical clustering methods, we divided the GBMs into two groups (n=19 and 37) that had significantly different survival lengths (p<0.05). In the two groups, expression of nine proteins (survivin, cyclin E, DCC, TGF-β, CDC25B, histone H1, p-EGFR, p-VEGFR2/3, p16) was significantly changed (q<0.05). Prognosis-predicting potential of these proteins were validated with another independent library of 82 GBM TMAs and a public GBM DNA microarray dataset. In addition, we determined 32 aberrant or mislocalized subcellular protein expression patterns in GBMs compared with relatively normal brain tissues, which could be useful for diagnostic biomarkers of GBM. We therefore suggest that these proteins can be used as predictive biomarkers and/or potential therapeutic targets for GBM.

Genomic instability and resistance to genotoxic therapies for glioblastoma (GBM) suggest aberrant DNA damage response (DDR), since DDR maintains the genomic integrity against genotoxic insults including anti-tumor therapies. To elucidate the biological and clinical meaning of DDR in GBM, we retrospectively investigated the immunohistochemical expression of DDR proteins (ATM, Chk1, Chk2, TopBP1, Rad17, p53, Nbs1, MDC1, γH2AX and RPA1) in 69 GBM surgical samples and their relation with GBM patient survival. Remarkably, higher expression of ATM revealed significantly longer overall survival (OS) and progression-free survival (PFS) (p<0.05). Upon multivariate analysis, expression level of ATM was an independent factor for longer OS (p=0.020) and longer PFS (p=0.019). Since ATM induces cell cycle arrest or apoptosis through cell cycle regulators in response to genotoxic insults, these results indicate that aberrant DDR signaling through ATM in GBM may be associated with resistance to genotoxic anti-tumor therapeutics. Conclusively, we emphasize that the identification of DDR machinery, which can be involved in unstable genomic status or genotoxic therapies in GBM, is very important to predict patient outcome.

The presence of active immunity within the brain supports the possibility of effective immunotherapy for glioblastoma (GBM). To provide a clinically-relevant adoptive immunotherapy for GBM using ex vivo expanded cytokine-induced killer (CIK) cells, the treatment capability of CIK cells, either alone or in combination with temozolomide (TMZ) were evaluated. Human CIK (hCIK) cells were cultured from PBMC using activating anti-CD3 antibody and IL-2, which were 99% CD3+, 91% CD3+CD8+ and 29% CD3+CD56+. In vitro, hCIK cells showed tumor-specific cytotoxicity against U-87MG human GBM cells. When hCIK cells were injected into tail veins of immune-compromised mice bearing U-87MG tumors in their brains, numerous CIK cells infiltrated into the brain tumors. CIK treatments (1x10(5), 1x10(6) or 1x10(7), once a week for four weeks) inhibited the tumor growth significantly in a dose-dependent manner; 44, 54 and 72% tumor volume reduction, respectively, compared with the control group (P<0.05). Moreover, hCIK cells (1x10(7), once a week for four weeks) and TMZ (2.5 mg/kg, daily for 5 days) combination treatment further increased tumor cell apoptosis and decreased tumor cell proliferation and vessel density (P<0.05), creating a more potent therapeutic effect (95% reduction in tumor volume) compared with either hCIK cells or TMZ single therapy (72% for both, P<0.05). Taken together, CIK cell-immunotherapy and TMZ chemotherapy have synergistic therapeutic effects and could be combined for a successful treatment of GBM.

Previous studies showed promise of coupling genetically engineered neural stem cells (NSCs) with blood-brain barrier permeable prodrugs as an effective anti-brain tumor therapy. Here, we further advance those findings by testing the suicide gene therapeutic system in syngenic glioblastoma immunocompetent mice. After intracranial injection of HB1.F3.CD, an immortalized human NSC cell line engineered to constitutively produce cytosine deaminase (CD), the prodrug 5-fluorocytosine (5-FC) was administered for five days, q.d., via intraperitoneal injection. The HB1. F3.CD hNSCs migrated specifically to the brain tumor site via the corpus callosum and significantly reduced the tumor volume (67%) by converting 5-FC into the cytotoxic 5-fluorouracil. A corresponding increase in F4/80-positive population was observed in the treatment group, although CD3-positive population remained unchanged compared to control. No toxic effects or morphological changes were observed in the spleen and the lymph nodes. The data suggest that the NSC-enzyme/prodrug treatment is an effective anti-tumor therapeutic strategy that specifically targets only the tumor site with little or no systemic side effects. In addition, the treatment modeled here successfully elicited a macrophagic immune response which seemed to have a synergistic role in reducing tumor volume, thus showing promise for treatment-mediated enhancement of inherent immune responses against brain tumors.

P-glycoprotein (P-gp), a factor responsible for the multidrug resistance of tumors, is specifically expressed in brain microenvironment. To test its roles in brain metastatic tumor chemoresistance, we implanted the paclitaxel-sensitive melanoma cell line, K1735, into the skin or brain of mice and examined its paclitaxel resistances. When implanted into the skin, paclitaxel inhibited tumor growth, however, it had no inhibitory effect on cells implanted into the brain. The paclitaxel resistance of the brain K1735 tumors was eliminated by combined treatment with a P-gp inhibitor, HM30181A, and paclitaxel. Previously we found that there is a defined therapeutic window for combined treatment of brain tumors with HM30181A and paclitaxel. To determine whether it is due to responses of the brain microenvironment we measured changes in P-gp expression and function of brain endothelial cells in response to HM30181A treatment in vitro and in vivo. They were significantly increased by high-dose HM30181A treatment and it was related with the therapeutic effect loss of high-dose HM30181A treatment. Therefore, P-gp in the brain microenvironment has crucial roles in the brain metastatic tumor chemoresistance and brain microenvironment responses to P-gp inhibitor treatment should be considered in the development of brain endothelial cell-targeted chemotherapy using P-gp inhibitor.

Since cancer stem cells in brain tumors were introduced, there have been few explanations regarding the role of cancer stem cells in the progression of glioma. Here, we investigated their major molecular changes in tumor progression in relation to the stem cell subpopulation. Using 12 surgical specimens of gliomatosis cerebri (GC) in the early and advanced stages, we measured the expression of a panel of cell proliferation, microvessel density, microvessel areas, angiogenic factors and their associated receptors. In addition, expression of neural stem cell markers and associated cytokines were examined in tumor tissues by quantitative real-time RT-PCR. Comparing the biological characteristics between the initial infiltrating lesions (n=7) and progressed lesions (n=5), Sox2 and Musashi-1 were expressed in the tumor tissue at an early and a progressed state. Contrary to the early infiltrative phase representing angiogenesis-independent growth, GC with progression showed that nestin (+), PCNA (+) cells and total vessel area (angioectasia) were markedly increased with a higher expression of proangiogenic molecules and their receptors. These results suggest that tumor progression is mediated by cancer stem cells and cross-talk of cancer stem cells along with their environment and are closely associated with angiogenesis-dependent progression and -independent growth.

Oral chemotherapy has many advantages over parenteral chemotherapeutics administration. To use the advantages of the oral chemotherapy and maximize anti-tumor effects of the chemotherapeutic agent, we designed HM30181A (a P-glycoprotein inhibitor) and a paclitaxel oral co-administration chemotherapeutic method. HM30181A is used to aid paclitaxel absorption from gut lumen into blood and to inhibit paclitaxel exclusion out of the brain tumor mass by endothelial cells, which inhibits paclitaxel access to tumor cells in the brain parenchyma. We applied HM30181A and paclitaxel oral co-administration methods to the treatment of tumors in the brain using the K1735 melanoma brain metastasis animal model and the U-87 MG glioblastoma animal model. Administrations were performed twice per week for 28 days and the therapeutic effect was examined using tumor volume change. We observed that 32 mg/kg HM30181A and 16 mg/kg of paclitaxel (dose ratio 2:1) oral co-administration showed significant therapeutic effects in both animal models, but when the doses or dose ratio was changed, the effects could not be observed. Therefore, adjustments of doses and dose ratio of the agents seems to be essential in realizing oral HM30181A and paclitaxel treatment in brain tumors. These results suggest that if the doses and dose ratio can be successfully adjusted, the oral co-administration of HM30181A and paclitaxel can be used to treat tumors in the brain.

Frequent regular administration of chemotherapeutic agents at low doses, known as 'metronomic chemotherapy', can increase the anti-angiogenic activity of the drugs, as has been confirmed by several other experimental tumor models. The aim of this pilot study was to evaluate the efficacy and safety of metronomic temozolomide (TMZ) treatment in twelve consecutive patients with recurrent TMZ-refractory glioblastoma. The patients were administered by metronomic treatment schedule (continuous low-dose chemotherapy) with TMZ at a daily dose of 40 mg/m(2). The median overall survival (OS) and progression-free survival (PFS) from the start of metronomic treatment were 11.0 months (95% CI, 5.2-10.5 months) and 6.0 months (95% CI, 0-12.3 months), respectively. During the follow-up period, complete response (CR) was not achieved in any patient, partial response (PR) in 2, and stable disease (SD) in 5 patients. Estimated PFS (CR+PR+SD) was 58.3% at 3 months. Grade III/IV toxicity according to the National Cancer Institute Common Toxicity Criteria (NCI CTC) was not found. These results suggest that the change of chemotherapeutic schedule from conventional to metronomic treatment overcomes the chemo-resistance in patients with recurrent TMZ-refractory glio-blastoma without any major toxicity.

The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo.

Glioblastoma is a highly angiogenic tumor with a dismal prognosis. Temozolomide (TMZ), a methylating agent is one of the most effective chemotherapeutic agents against glioblastoma. To overcome the problem that most of these tumors become resistant to chemotherapeutic regimens within a year, we investigated the antitumor efficacy of metronomic administration of low-dose TMZ in in vitro cell proliferation/cytotoxicity assay and in vivo rat and nude mouse orthotopic glioma model. By in vitro assay, we elucidated that C6/LacZ rat glioma cells were more resistant to metronomic treatment of TMZ than U-87MG human glioblastoma cells and bEnd.3 mouse brain endothelial cells. Compared with the conventional chemotherapeutic regimen of TMZ, we found that frequent administration of TMZ at a low dose (metronomic treatment) markedly inhibited angiogenesis as well as tumor growth in a TMZ-resistant C6/LacZ rat glioma model. In addition, metronomic treatment of TMZ significantly augmented apoptosis of tumor cells in this model. For the TMZ-sensitive U-87MG cells, even with a very low dose of TMZ, which is not effective to reduce tumor mass, the metronomic treatment of TMZ reduced the microvessel density, i.e. angiogenesis, in a nude mouse orthotopic model. In conclusion, for both models, the metronomic treatment of TMZ decreased angiogenesis. Especially, in TMZ-resistant glioma cells, this regimen increased apoptosis of tumor cells and decreased tumor growth. The metronomic treatment of TMZ in orthotopic glioma models demonstrated a successful antiangiogenic effect which can overcome the chemoresistance in conventional TMZ chemotherapy.

Metastatic breast cancer is still defined as an incurable disease, with the lungs being the most common metastatic sites in breast cancer patients. Epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, is known to be involved in survival, migration, angiogenesis and metastasis of cancer. The spontaneous pulmonary metastasis mouse model was applied to evaluate the effects of the EGFR tyrosine kinase inhibitor, erlotinib, on the prevention of pulmonary metastasis in curatively resected breast carcinoma. The expression of EGF and EGFR was significantly strong in pulmonary metastatic nodules compared to those in primary breast carcinoma tissue. A treatment of erlotinib (oral gavage, 50 mg/kg/day, every day for 6 weeks) given to mastectomized mice inhibited the incidence of pulmonary metastasis. The number of metastatic pulmonary nodules was significantly reduced in the erlotinib-treated group compared with the control. Therefore, erlotinib may play a role in preventing pulmonary metastasis, which shows the strong expression of EGF and EGFR after curative resection of primary breast cancer.

Glioblastomas are highly vascularized tumors and anti-angiogenic strategy is one of the most promising therapeutic approaches to treat brain tumors. Interferon alpha (IFN-alpha) as a single agent or combined with standard chemo-therapy has been shown to inhibit various tumors, but the effect of combination anti-angiogenic therapy on brain tumors has not been well studied. We determined the optimal dose and schedule of pegylated IFN-alpha (PEG-IFN-alpha) against U-87MG human glioblastoma cells growing orthotopically in nude mice, since several clinical trials reported that PEG-IFN-alpha administered at higher or lower doses was less effective. The group treated two times per week with injections of 10 KU of PEG-IFN-alpha for 4 weeks showed significant decreases in cell proliferation and angiogenesis. Moreover, the optimal dose and schedule of PEG-IFN-alpha determined in this study and combined with paclitaxel treatment potently inhibited tumor growth in vivo. The mechanisms of the significant therapeutic effects were most likely caused by directly inhibiting cell proliferation and angiogenesis, and rendering apoptosis increased. Specifically PEG-IFN-alpha/paclitaxel combination induced apoptosis of tumor-associated endothelial cells more than that of tumor cells. These results suggest that optimal biological dosage and scheduling of PEG-IFN-alpha and paclitaxel combination is a potent strategy for glioblastoma patients as a new synergistic anti-endothelial treatment.

Gliomas are primary brain tumors associated with a poor prognosis partly due to resistance to conventional therapies. To overcome this problem, we investigated the combined effects of gamma knife radiosurgery (GKS) and an antiangiogenic agent, thalidomide (THD), or a chemotherapeutic agent, temozolomide (TMZ), on a rat glioma model. GKS (20 Gy single dose) alone and/or drugs (for 3 days) were delivered 14 or 18 days after stereotactic implantation of C6/LacZ glioma cells into the brains of Sprague-Dawley rats. A group of animals treated with or without drugs for 3 days was irradiated on day 18 and sacrificed at 24 h after GKS to evaluate cell proliferation, apoptosis and microvessel density. The other group of animals was irradiated on day 14 and sacrificed at day 5 after GKS for the measurement of tumor volume. Apoptosis of endothelial cells in the tumor beds was only observed in the early period after GKS. Decreased cell proliferation and increased tumor cell apoptosis were observed in rat gliomas treated with GKS and THD or TMZ. The combination treatments with GKS and THD or GKS and TMZ also decreased microvessel density, i.e. angiogenesis, more effectively compared with GKS treatment alone. The combination of GKS and THD was the most effective regimen, resulting in a significant decrease of tumor volume. We suggest that the antitumor effect of GKS on glioma is enhanced by the addition of THD. Therefore, combined therapy with GKS and THD might be a favorable treatment for gliomas.

The chemotherapeutic agent temozolomide (TMZ) and the anti-angiogenic agent thalidomide (THD) have both demonstrated anti-tumor activity in patients with recurrent malignant glioma. Combination treatment with TMZ and THD in patients with glioblastoma multiforme (GBM) appears to be more effective than treatment with either drug alone. To investigate the mechanism of this anti-tumor effect, we examined the combined effects of TMZ and THD in a rat glioma xenograft model. We found that combination treatment markedly inhibited the growth of tumors that were orthotopically implanted into rat brains. Using proliferating cell nuclear antigen (PCNA) staining, we observed a significant decrease in cell proliferation in these tumors. CD31 staining of the microvasculature revealed a significant decrease in angiogenesis. We also found increased apoptosis in treated tumors by terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. We further demonstrated that the expression of angiogenic factors, such as vascular endothelial cell growth factor (VEGF) and basic fibroblastic growth factor (bFGF), were inhibited by THD. THD also decreased the number of ED1-positive, activated macrophages or microglial cells, which produce pro-angiogenic molecules around the glioma. Taken together, these results suggest that combination treatment with TMZ and THD inhibits tumor growth via the induction of apoptosis and the inhibition of angiogenesis in a rat model and may be a promising therapy for malignant gliomas.

The purpose of this study was to determine whether a combination treatment of temozolomide with celecoxib is effective in the rat orthotopic glioma model. After stereotactic injection of C6/LacZ rat glioma cells into the Sprague Dawley rat brain, the rats were randomly assigned to four treatment groups [group 1, control treatment; group 2, celecoxib (25 mg/kg p.o. everyday) alone; group 3, temozolomide (7.5 mg/kg i.p. for 5 days at 2nd week) alone; group 4, a combination of celecoxib and temozolomide]. Rats were sacrificed 18 days after treatment, and the body weight, tumor volume, tumor cell proliferation, microvessel densities, and apoptosis were evaluated. There was a significant reduction of tumor volume in combination group compared to control or single-agent therapy. The median tumor volume was estimated to be 111.5 mm(3) (control), 65.0 mm(3) (celecoxib), 71.8 mm(3) (temozolomide) and 18.7 mm(3) (combination). In the combination group, there was increased tumor cell apoptosis as well as decreased microvessel density and tumor cell proliferation relative to the control and single-agent therapy (P<0.05). Collectively, the data suggest that the combination celecoxib and temozolomide may provide a novel and effective approach to the treatment of glioblastoma.

Brain specific angiogenesis inhibitor (BAI)-1 is a novel p53-inducible anti-angiogenic molecule. We examined the expression of BAI-1 in glial tumors and its association with patient survival. The expression of BAI-1 was evaluated in 20 brain tumors (meningiomas, pituitary adenomas, hemangiopericytomas, hemangioblastomas), 2 normal brain samples, 5 benign gliomas, and 26 glioblastomas. In the 26 glioblastoma tumors, we also evaluated the expression of VEGF, p53, p53 mutations, and MIB-1 to determine their association with survival. BAI-1 mRNA was expressed in all benign gliomas, normal brain, and 9 out of 26 glioblastomas, but not in the other tumors. Low VEGF and aberrant high expression of p53 were associated with a favorable outcome in univariate survival analysis, but they were not independent factors in multivariate analysis. For the treatment response, BAI-1 expression was associated with better response to radiation therapy (p=0.014). When we divided the patients into groups according to the expression patterns of BAI-1 and VEGF mRNA, the median survival of 9 patients with high VEGF expression and no expression of BAI-1 was just 6 months, while the median survival of the other 17 patients was 14 months (p=0.013). Glioblastomas with no BAI-1 and high VEGF mRNA expression are more often associated with poor clinical outcome. These findings suggest that the balance between the angiogenic and anti-angiogenic factors is important in the progression of glioblastoma and its response to treatment.

Higher cyclooxygenase-2 (COX-2) expression is clinically associated with more aggressive gliomas and is a strong predictor of poor survival. To determine whether oral administration of a COX-2-specific inhibitor can inhibit glial tumors, we analyzed the effect of celecoxib on the growth of 9L rat gliosarcoma cells that were orthotopically transplanted into rat brains. Oral administration of celecoxib beginning 1 day after implantation of 5 x 10(4) 9L rat gliosarcoma cells into rat brain reduced the incidence and size of tumors significantly. Immunohistochemical analysis of implanted gliosarcoma cells from rats treated with celecoxib showed lower levels of phospho-Akt, phospho-EGFR, Bcl-2, and Bcl-XL expression compared with untreated tumor cells. Gliosarcoma cells from treated rats had significantly more TUNEL- and caspase-3-positive cells and fewer PCNA-positive cells. These results demonstrate that selective COX-2 inhibitors may be useful as adjuvants and/or therapeutic agents to treat gliomas overexpressing COX-2.

Using a microdissection technique, the contribution of the p53 mutation to tumorigenesis and prognosis in each histological subtype of the intracranial germ cell tumors (GCTs) was evaluated. Nineteen patients had primary intracranial GCTs, including 4 germinomas (GEs), 4 teratomas (TEs), 1 mixed tumor of GE and TE, and 10 mixed GCTs containing non-germinomatous malignant germ cell tumors (NG-MGCTs). After microdissection of specific subtypes, genomic DNA was screened for mutations in exons 5-8 of the p53 gene, using the dideoxyfingerprinting (ddF) followed by direct DNA sequencing. The direct sequencing revealed a total of six mutations in PCR products derived from the five cases (26%) which showed mobility shifts in ddF. Among the six mutations detected, four were missense mutations and two were silent. Missense mutations of the p53 gene tended to occur more frequently in the NG-MGCT component than in the GE or TE components (3/15 vs. 1/12 vs. 0/13). The incidence of missense mutations was not different between the survivors (3/13) and the deceased (1/6). This study suggests the possible role of the p53 gene in the tumori-genesis of NG-MGCT. However, p53 gene mutation did not correlate with the prognosis of NG-MGCT.