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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-XL 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-XL 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. © 2001 Wiley-Liss, Inc.

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.

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 HSVtkgene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtkgene 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 HSVtkgene (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.

Bax-mediated apoptosis in neurons is involved in many pathologic conditions affecting the central nervous system, including degenerative diseases, stroke, and trauma. Two molecules belonging to the Bcl-2 family, Bcl-2 and Bcl-XL, protect cells from Bax-induced apoptosis and show distinct expression patterns in adult neurons, with downregulated Bcl-2 and highly upregulated Bcl-XLexpression. To investigate the biological functions of these two molecules in Bax-mediated apoptosis in neurons, we transduced various levels of Bcl-XLor Bcl-2 via adenoviral vectors into nerve growth factor (NGF)-treated PC12 cells. Overexpression of Bax induced drastic apoptosis in NGF-treated PC12 cells. Bcl-XLexpressed at a wide range of levels conferred a high level of protection against Bax-mediated apoptosis. In contrast, Bcl-2 at various levels conferred far less protection against apoptosis. Moreover, Bcl-XLprotected PC12 cells from apoptosis induced by NGF withdrawal. These data indicate that Bcl-XL-mediated protection is the major pathway that suppresses apoptosis in NGF-treated PC12 cells and that Bcl-XLwould be a more relevant target of manipulation in future treatment strategies, including gene therapies.

Tumor necrosis factor‐α (TNF), which was initially supposed to be a promising cancer therapeutic reagent, does not kill most types of cancer cells partly due to the activation of an anti‐apoptotic gene, NF‐kB. NF‐kB forms an inactive complex with the inhibitor kappa B alpha (IkBα), which is rapidly phosphorylated and degraded in response to various extracellular signals. To disrupt this protective mechanism, we introduced an inhibitor kappa B alpha (IkBdN)gene, a deletion mutant gene lacking the nucleotides for the N‐terminal 36 amino acids of IkBα, into human glioma cells (U251, T‐98G, and U‐373MG) via an adenoviral (Adv) vector in addition to treatment of the glioma cells with recombinant TNF. Immunohistochemical analysis revealed that NF‐kB was translocated to nuclei by TNF treatment in U251 and T‐98G cells, but not in U‐373MG cells. Neither transduction of IkBdN nor treatment with TNF protein alone induced apoptosis in U251 and T‐98G cells, whereas both cell lines underwent drastic TNF‐induced apoptosis after transduction of IkBdN. On the other hand, U‐373MG cells were refractory to TNF‐induced apoptosis even when they were transduced with the IkBdNgene. U‐373MG cells underwent drastically increased apoptosis when co‐transduced with the IkBdNand Baxgene in the presence of TNF. Adv‐mediated transfer of IkBdN or IkBdN plus Bax may be a promising therapeutic approach to treat gliomas through TNF‐mediated apoptosis.

A case of multiple intracranial germ cell tumor in which a pineal tumor regressed spontaneously after resection of the cerebellar mass is reported. Immunohistochemical staining of the cerebellar mass showed that most of the infiltrating lymphocytes were positive for CD3 and CD8. The anti-Ki-67 monoclonal antibody MIB-1 staining of the resected tumor revealed a high MIB-1 positivity ratio (36.1%) among the large tumor cells, and TUNEL staining demonstrated that positivity in up to 6% of the tumor cells. Possible mechanisms responsible for this spontaneous regression including immunological responses and apoptosis induced by T lymphocytes are discussed.

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 β-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.