Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Co-transduction of Apaf-1 and caspase-9 highly enhances p53-mediated apoptosis in gliomas.

Mutation of the p53 gene plays a critical role in the development of cancer and response to cancer therapy. To analyze the mechanism of cancer development and to improve cancer therapy, it is important to assess which genes are downstream components of p53 in cancers, and whether the expression levels of these genes affect p53-mediated apoptosis. In this study, we transduced the wild type p53 gene along with the Apaf-1 and caspase-9 genes via adenovirus vectors into U251 and U-373MG glioma cells harbouring a mutated p53, and evaluated the degree of apoptosis. Co-induction of Apaf-1 and caspase-9 genes highly enhanced p53-mediated apoptosis in glioma cells. Induction of wild type p53 enhanced the expression levels of Bax, p21/WAF1, and Fas protein. To determine which gene is activated by wild type p53 induction and, in turn, activates Apaf-1 and caspase-9, we transduced the Bax, p21/WAF1 or Fas gene via adenovirus vector to U251 cells to achieve a similar expression level as that induced by the Adv for p53 in U251 cells. U251 cells transduced with Fas concomitant with the Apaf-1 and caspase-9 genes underwent drastic apoptosis. This suggests that induction of wild type p53 upregulates Fas, which in turn may play a role in the activation of Apaf-1 and caspase-9. These results are important for analyzing the mechanism of tumour development and for predicting the therapeutic effect of p53 replacement gene therapy in a particular patient.

Dietary curcuminoids prevent high-fat diet-induced lipid accumulation in rat liver and epididymal adipose tissue.

Curcumin and its structurally related compounds (curcuminoids), the phenolic yellowish pigments of turmeric, display antioxidative, anticarcinogenic and hypocholesterolemic activities. In this study, we investigated the effects of dietary supplemented curcuminoids [commercial grade curcumin: a mixture of curcumin (73.4%), demethoxycurcumin (16.1%) and bisdemethoxycurcumin (10.5%)] on lipid metabolism in rats. Male Sprague-Dawley rats were assigned to three diet groups (n = 6) and fed a moderately high-fat diet (15 g soybean oil/100 g diet) for 2 wk. One diet group did not receive supplements (CONT), while the others were supplemented with 0.2 g curcuminoids/100 g diet (CUR0.2) or 1.0 g curcuminoids/100 g diet (CUR1.0). Liver triacylglycerol and cholesterol concentrations were significantly lower in CUR1.0 rats than in CONT rats. Plasma triacylglycerols in the VLDL fraction were also lower in CUR1.0 rats than in CONT rats (P < 0.05). Hepatic acyl-CoA oxidase activity of both the CUR0.2 and CUR1.0 rats was significantly higher than that of CONT rats. Furthermore, epididymal adipose tissue weight was significantly reduced with curcuminoid intake in a dose-dependent manner. These results indicate that dietary curcuminoids have lipid-lowering potency in vivo, probably due to alterations in fatty acid metabolism.

Doctors' and nurses' attitudes towards and experiences of voluntary euthanasia: survey of members of the Japanese Association of Palliative Medicine.

OBJECTIVE: To demonstrate Japanese doctors' and nurses' attitudes towards and practices of voluntary euthanasia (VE) and to compare their attitudes and practices in this regard. DESIGN: Postal survey, conducted between October and December 1999, using a self-administered questionnaire. PARTCIPANTS: All doctor members and nurse members of the Japanese Association of Palliative Medicine. MAIN OUTCOME MEASURE: Doctors' and nurses' attitude towards and practices of VE. RESULTS: We received 366 completed questionnaires from 642 doctors surveyed (response rate, 58%) and 145 from 217 nurses surveyed (68%). A total of 54% (95% confidence interval (CI): 49-59) of the responding doctors and 53% (CI: 45-61) of the responding nurses had been asked by patients to hasten death, of whom 5% (CI: 2-8) of the former and none of the latter had taken active steps to bring about death. Although 88% (CI: 83-92) of the doctors and 85% (CI: 77-93) of the nurses answered that a patient's request to hasten death can sometimes be rational, only 33% (CI: 28-38) and 23% (CI: 16-30) respectively regarded VE as ethically right and 22% (CI: 18-36) and 15% (CI: 8-20) respectively would practise VE if it were legal. Logistic regression model analysis showed that the respondents' profession was not a statistically independent factor predicting his or her response to any question regarding attitudes towards VE. CONCLUSIONS: A minority of responding doctors and nurses thought VE was ethically or legally acceptable. There seems no significant difference in attitudes towards VE between the doctors and nurses. However, only doctors had practised VE.

Caspase-9 transduction overrides the resistance mechanism against p53-mediated apoptosis in U-87MG glioma cells.

OBJECTIVE: Conflicting reports have been published with regard to the relationship between the efficacy of p53 gene therapy and the p53 status of gliomas. In this study, we evaluated whether U-87MG glioma cells harboring wild-type p53 and U251 and U-373MG glioma cells harboring mutated p53 demonstrate different sensitivities to p53-induced apoptosis. In addition, we tested whether transduction of Bax or caspase-9, which are downstream components of p53-induced apoptosis, can override the resistance mechanism of U-87MG cells to apoptosis. METHODS: We transduced U-87MG, U251, and U-373MG glioma cells with p53, Bax, or caspase-9 genes via adenovirus (Adv) vectors, to induce the same level of respective proteins, and evaluated the degree of apoptosis. RESULTS: U-87MG cells were highly resistant to Adv for p53 (Adv-p53)-mediated apoptosis, whereas U251 and U-373 cells underwent extensive apoptosis after Adv-p53 infection. In U-87MG cells, the elevation of Bax and Fas was not as marked as that observed in U251 and U-373MG cells after Adv-p53 infection. Endogenous expression of Bcl-XL and Bcl-2 in U-87MG cells was greater than that in U251 and U-373MG cells. U-87MG cells were more resistant to Bax-mediated apoptosis than were U251 or U-373MG cells. In contrast, U-87MG cells were more sensitive to caspase-9-mediated apoptosis than were U251 or U-373MG cells, suggesting that transduction of caspase-9 may override the resistance mechanism of U-87MG to p53-mediated apoptosis. CONCLUSION: These results demonstrate that proapoptotic function induced by p53 transduction in U-87MG cells was repressed at several steps and that induction of caspase-9 may circumvent this resistance mechanism.

Over-expression of APAF-1 and caspase-9 augments radiation-induced apoptosis in U-373MG glioma cells.

The p53 tumor-suppressor gene plays a critical role in radiation-induced apoptosis. Several genes, including Bax and Fas, are involved in p53-mediated apoptosis, and their over-expression enhances the degree of radiation-induced apoptosis. Apaf-1 and caspase-9 have been reported to be downstream components of p53-mediated apoptosis, suggesting that these genes play a role in radiation-induced apoptosis. In this study, we transduced U-373MG cells harboring mutant p53 with the Apaf-1 and/or caspase-9 genes via adenoviral (Adv) vectors concomitant with X-ray irradiation and evaluated the degree of apoptosis. The percentage of apoptotic cells in U-373MG cells co-infected with the Adv for Apaf-1 (Adv-APAF-1) and that for caspase-9 (Adv-Casp9) and treated with irradiation (24%) was much higher than that in cells co-infected with Adv-APAF-1 and Adv-Casp9 and not treated with irradiation (0.86%) and that in cells infected with either Adv-APAF-1 or Adv-Casp9 and treated with irradiation (2.0% or 2.6%, respectively). The apoptosis induced by co-transduction of Apaf-1 and caspase-9 and irradiation was repressed in cells that were co-infected with the Adv for Bcl-X(L) but not in cells co-infected with the Adv for Bcl-2. These results indicate that Apaf-1 and caspase-9 play a role in radiation-induced apoptosis in cancer cells harboring mutant p53. Bcl-X(L) may be critically involved in the radioresistance of cancer cells by repressing Apaf-1- and caspase-9-mediated apoptosis. Expression of Apaf-1 and caspase-9 in tumors may be an important determinant of the therapeutic effect of irradiation in cancer treatment.

Recurrence of infantile supratentorial ependymoma after 23-year remission following surgical removal and radiation therapy.

We report on a patient with ependymoma who had a recurrence after long-term remission. The patient developed frontoparietal ependymoma at the age of one year and ten months. The tumor was radically removed and postoperative radiation therapy was performed. A calcified area adjacent to the area of surgical removal remained unchanged until the patient was 18 years old. The patient was healthy except for mild hemiparesis until an MRI scan performed when he was 25 years old showed regrowth of the tumor. The patient underwent surgery with additional radiation therapy and was discharged. The 23-year interval until tumor recurrence in this case is far beyond the so-called risk period of "Collins' law". Immunohistochemical study with MIB-1 and anti-p53 antibody showed a high proliferative potential of the primary and recurrent tumors and possible p53 mutation in the primary tumor. This is the first report to describe the detailed clinical course and histological features of a recurrent infantile ependymoma that progressed after Collins' risk period. It seems that follow-up of ependymoma patients after initial treatment should be performed regularly for a longer period in cases showing radiological evidence of a residual lesion.

Dialysis patients' preferences regarding cardiopulmonary resuscitation and withdrawal of dialysis in Japan.

The aim of this study is to show the preferences of Japanese dialysis patients for receiving cardiopulmonary resuscitation (CPR) in their current health status, if they were severely demented, or if they had terminal cancer and to determine their desires about continuing dialysis if they were severely demented or had terminal cancer. A questionnaire survey including the three scenarios was administered to 450 dialysis patients in 15 hospitals in JAPAN: Three hundred ninety-eight patients completed the questionnaires for a response rate of 88%. The majority of responding patients were men and were undergoing hemodialysis. Only 5% of the patients had discussed their preferences regarding CPR with their physicians, and 29%, with their family members. Forty-two percent of the patients answered that they would want to receive CPR if they experienced cardiopulmonary arrest in their current health status, and 12% answered in the affirmative if they were seriously demented or had terminal cancer. Eighteen percent of the patients would want to continue dialysis if they were demented, and 45%, if they had terminal cancer. Statistical analysis showed that more patients who were working tended to want to continue dialysis if they had terminal cancer than those who were not (53% versus 37%; P < 0.014). Patients' age and preferences did not statistically correlate. Preferences of Japanese dialysis patients for CPR and dialysis vary according to differences in health status, and only a minority would want to receive CPR for cardiopulmonary arrest even in their current health status.

Adenovirus-mediated transfer of p53 augments hyperthermia-induced apoptosis in U251 glioma cells.

PURPOSE: Hyperthermia kills glioma cells by inducing apoptosis and is thereby an effective therapeutic modality for the treatment of malignant gliomas. However, cells harboring mutated p53 are refractory to hyperthermia-induced apoptosis. In this study, we assessed whether or not adenovirus (Adv)-mediated transduction of p53 overrides this resistant mechanism. METHODS AND MATERIALS: We transduced the p53 wild-type tumor suppressor gene into U251 glioma cells harboring mutated p53 using Adv vectors in combination with hyperthermia (43, 44.5 degrees C), and evaluated the degree of cell death and apoptosis. RESULTS: The percentage of cells that had died, as measured by trypan blue staining, among U251 cells infected with the Adv for p53 (Adv-p53) and treated with hyperthermia, was significantly higher than the percentage of cells that had died among U251 cells infected with Adv-p53 and not treated with hyperthermia, or those infected with the control Adv for dE (Adv-dE) and treated with hyperthermia. The degree of apoptosis, measured at 24 h after treatment, in hyperthermia-treated U251 cells infected with Adv-p53 (43 degrees C, 73%; 44.5 degrees C, 92%) was much higher than that infected with Adv-p53 (41%), or that infected with control Adv-dE and treated with hyperthermia (43 degrees C, 1.3%; 44.5 degrees C, 19%). Treatment with combined hyperthermia and Adv-p53 infection induced cleavage of caspase-3 in U251 cells. CONCLUSION: These results indicate that Adv-mediated transduction of p53 would render glioma cells highly sensitive to hyperthermia.

Co-transduction of Apaf-1 and caspase-9 augments etoposide-induced apoptosis in U-373MG glioma cells.

Several apoptosis-related genes have been reported to be involved in chemotherapy-induced apoptosis in cancers. An assessment of the relationship between expression of those genes and the degree of chemotherapy-induced apoptosis may be useful in improving the efficacy of cancer therapy. We transduced Apaf-1 (apoptotic protease-activating factor-1) and caspase-9 into U-373MG glioma cells using adenovirus (Adv) vectors in the presence of etoposide and evaluated the degree of apoptosis. The degree of apoptosis in etoposide-treated U-373MG cells infected with Adv for Apaf-1 (Adv-APAF1) was higher (27%) than that in cells infected with control Adv (14%), that in cells infected with Adv for caspase-9 (Adv-Casp9) was higher (34%) than that in cells infected with Adv-APAF1, and that in cells infected with both Adv-APAF1 and Adv-Casp9 was the highest (41%). Treatment with etoposide increased expression of p53 and decreased expression of Bcl-X(L) in U-373MG cells which harbored mutant p53. These results indicate that the expression of Apaf-1 and caspase-9 may be important determinants in predicting the sensitivity of cancers to chemotherapy. Adv-mediated co-transduction of Apaf-1 and caspase-9 should render cancer cells highly sensitive to chemotherapy.

Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line.

BACKGROUND: Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types. RESULTS: We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice. CONCLUSIONS: The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.

Radiation-induced apoptosis of oligodendrocytes in the adult rat brain.

Although radiation injuries to the brain are well documented, immediate early histological changes in the brain remain to be defined. The present study characterizes glial injury provoked in adult rat white matter within 24 h after a single irradiation of the whole brain (10 or 20 Gray). Irradiated brains were histologically and histochemically analyzed. TUNEL-positive cells exhibiting apoptotic morphology were counted in five representative regions of the white matter. Glial cell death was further evaluated by glial cell density 24 h after irradiation, which induced both dose (p < 0.0001)- and time- (p < 0.0001) dependent apoptosis in these cells. The overall apoptotic rate in the white matter peaked within 8 h after irradiation. Total glial cell density decreased significantly in the white matter 24 h after irradiation. TUNEL-positive cells were immunohistochemically negative for GFAP, a marker for astrocytes, but positive for CNP, a marker for oligodendrocytes. The apoptotic rate was highest in the external capsule (p < 0.0001), followed by the fimbria and genu of the corpus callosum (p < 0.0001). The rates were lowest in the internal capsule and cerebellum. These data indicated that brain irradiation induces rapid apoptotic depletion of the oligodendroglial population, which may participate in the development of radiation-induced pathological conditions.

Co-transduction of p27Kip1 strongly augments Fas ligand- and caspase-8-mediated apoptosis in U-373MG glioma cells.

BACKGROUND: p27Kip1 is a potential tumor suppressor gene. As malignant gliomas express Fas at high levels, the relationship between Fas-mediated apoptosis and p27Kip1 expression may improve therapeutic approaches for treating gliomas. MATERIALS AND METHODS: In this study, we transduced U-373MG glioma cells with the Fas ligand or caspase-8 genes using adenovirus vectors after transduction of the p27Kip1 gene to induce cell cycle arrest in U-373MG cells, and evaluated the degree of apoptosis. RESULTS: The results demonstrate that expression of p27Kip1 enhanced Fas ligand- or caspase-8-mediated apoptosis in U-373MG cells. Expression of apoptosis-related genes such as Bax, Bcl-X(L), Bcl-2 or caspase-8 were reduced by p27Kip1 transduction compared with that of beta-actin, whereas p27Kip1 transduction did not affect the expression level of Fas or the Fas ligand. CONCLUSION: Combined transduction of p27Kip1 with Fas ligand or caspase-8 would overide the resistance mechanism to apoptosis in malignant gliomas.

Degree of apoptosis induced by adenovirus-mediated transduction of p53 or p73alpha depends on the p53 status of glioma cells.

It has been reported that U-87MG glioma cells with wild-type p53 are resistant to p53 replacement gene therapy. As some gliomas harbor wild-type p53, it would be important to override the resistance mechanism due to wild-type p53 in glioma gene therapy. In this study, we transduced U-87MG cells or U251 glioma cells harboring mutated p53 with the p53 or p73alpha gene (a homologue of p53, that differently induces some p53-responsive genes) via adenovirus vectors (Advs) at same multiplicities of infection (MOIs) into respective cells (U-87MG: MOI 1000, U251: MOI 100), and evaluated the degree of apoptosis. The results demonstrate that the degree of apoptosis induced by Adv-mediated transduction of p53 in U-87MG cells was lower than that in U251 cells, whereas that induced by Adv-mediated transduction of p73alpha in U-87MG cells was higher than that in U251 cells. Bax expression in U-87MG and U251 cells induced by Adv-mediated transduction of p53 was almost the same as that of p73alpha. On the other hand, Adv-mediated transduction of p73alpha induced caspase-9 at higher levels than that of p53 in both cells. The results indicate that Adv-mediated transduction of p73alpha might be beneficial to overcome the resistance mechanism of glioma cells harboring wild-type p53.

Transduction of a fiber-mutant adenovirus for the HSVtk gene highly augments the cytopathic effect towards gliomas.

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk) / ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U-87MG and U-373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F / K20, which has a stretch of 20 lysine residues added at the C-terminus of the fiber, for the HSVtk gene (Adv-TK-F / K20), and compared the cytopathic effect of Adv-TK-F / K20 with that of the Adv for HSVtk with wild-type fiber (Adv-TK). The cytopathic effect of Adv-TK-F / K20 in U-87MG and U-373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv-TK. At the same multiplicity of infection (MOI) in each cell line, Adv-TK-F / K20 induced a higher degree of apoptosis (U-87MG, 35%; U-373MG, 77%) than Adv-TK (U-87MG, 0.11%; U-373MG, 27%) in U-87MG (MOI 0.03) and U-373MG cells (MOI 0.1). Cleavage of poly(ADP-ribose)polymerase (PARP) was more marked in the cells that were infected with Adv-TK-F / K20 than in cells that were infected with Adv-TK. These results indicate that gene therapy utilizing Adv-TK-F / K20 may be a promising therapeutic modality for the treatment of gliomas.

Adenovirus-mediated transfer of Fas ligand gene augments radiation-induced apoptosis in U-373MG glioma cells.

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.

Levels of beta-human chorionic gonadotropin in cerebrospinal fluid of patients with malignant germ cell tumor can be used to detect early recurrence and monitor the response to treatment.

BACKGROUND: Tumor marker-producing germ cell tumors of the central nervous system are malignant and require radiation and/or chemotherapy. Although serum beta-human chorionic gonadotropin (hCG) has been used to monitor the course of treatment, the levels of beta-hCG in the cerebrospinal fluid (CSF) have not been measured routinely in the clinic. To determine whether they can be used to evaluate parameters of tumor status, such as progression or response to therapy, levels of beta-hCG in the serum and CSF of patients with germ cell tumors were studied. METHODS: Fifty-four paired samples of CSF and serum were taken from seven patients with germ cell tumor and their beta-hCG levels were measured. Beta-hCG was negative in both serum and CSF in 11 instances and the levels in the other 43 paired samples were analyzed for any correlation or relationship to therapy. They were also compared with the clinical courses. RESULTS: The mean CSF beta-hCG level was 11.5 mIU/ml, which was significantly higher than the level in serum (3.5, p = 0.002). In all the paired samples except for one time point, the level in CSF was higher than that in serum. Out of 43 instances where the beta-hCG level in CSF was elevated, the level in serum was elevated in only 16 (37.2%). Among cases of recurrent malignant germ cell tumor, there were nine instances of recurrence or progression despite therapy. In all five instances where beta-hCG CSF levels were measured, the levels were elevated prior to any increase or detectability of the serum values. CONCLUSION: It seems likely that the level of beta-hCG in CSF is a good marker for monitoring tumor recurrence or evaluation of treatment results.

Adenovirus-mediated transfer of p53 and Fas ligand drastically enhances apoptosis in gliomas.

Various therapeutic approaches toward killing glioma cells by inducing apoptosis have been developed, but these approaches are often hampered by anti-apoptotic mechanisms. In this study, we attempted to develop a technique that overrides the resistance toward apoptosis in glioma cells. To date, p53- and Fas-mediated apoptotic pathways have been shown to be different. Therefore, we carried out a gene therapy that combines the pro-apoptotic effect of these two different pathways. The recombinant adenoviruses (Advs) for p53 and Fas ligand (FL) (Adv-p53 and Adv-FL, respectively) were constructed. Transfecting the p53 gene into glioma cell lines (A-172 and U251 glioma cells) led to overexpression of Bax, a protein that induces permeability transition; at the same time, this transfection brought about an overexpression of Fas. To intensify Fas-mediated apoptosis, we transferred the FL gene together with the p53 gene by Adv-mediated gene transduction into A-172 and U251 cells. Coinfecting Adv-p53 and Adv-FL into A-172 cells, which are relatively resistant to apoptosis by infection with Adv-p53 or Adv-FL alone (Adv-p53, multiplicity of infection (MOI) of 100: 8.5 +/- 0.7%; Adv-FL, MOI of 100: 3.0 +/- 0.1%) resulted in a drastic enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 24.2 +/- 0.9%). Coinfection with Adv-p53 and Adv-FL in U251 cells resulted in a similar enhancement of the percentage of apoptotic cells (Adv-p53 and Adv-FL, each at an MOI of 30: 59.0 +/- 2.3%) compared with that seen in U251 cells transfected with Adv-p53 or Adv-FL alone (Adv-p53, MOI of 30: 3.1 +/- 0.3%; Adv-FL, MOI of 30: 18.1 +/- 0.3%). Regardless of whether a cell line is resistant or sensitive to FL- and p53-mediated apoptosis, coinfection with Adv-p53 and Adv-FL dramatically enhanced the degree of apoptosis of glioma cells. Our results indicate that the coinfection approach can be used as a modality for the gene therapy of gliomas, overriding the apoptosis-resistant mechanisms in glioma cells.