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.

A report on small team clinical ethics consultation programmes in Japan.

Clinical ethics support, including ethics consultation, has become established in the field of medical practice throughout the world. This practice has been regarded as useful, most notably in the UK and the USA, in solving ethical problems encountered by both medical practitioners and those who receive medical treatment. In Japan, however, few services are available to respond to everyday clinical ethical issues, although a variety of difficult ethical problems arise daily in the medical field: termination of life support, euthanasia and questions about patient autonomy. In light of these conditions, a group of 17 volunteer educators and researchers from the area of biomedical ethics, including the authors, have formed the Clinical Ethics Support and Education Project, and began providing Japan's first small team clinical ethics consultation service in October, 2006. Members include scholars of biomedical ethics, scholars of philosophy and ethics, legal professionals and legal scholars, nurses and doctors, consisting of five women and 12 men. Consultation teams, made up of a small number of members, were organised each time a request for consultation was received. Over approximately 15 months (October 2006-December 2007), the programme received 22 consultation requests from medical practitioners and medical institutions, and three from the families of patients. In this paper, we will discuss the status of our consultation service and examples of consultation cases we have handled. In addition, we will examine the process of evaluating small team clinical ethics consultation services, as well as the strengths and weakness of such programmes.

A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis.

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 beta-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.

Characterization of a duocarmycin-DNA adduct-recognizing protein in cancer cells.

Duocarmycins have been reported to derive their potent antitumor activity through a sequence-selective minor groove alkylation of N3 adenine in double-stranded DNA. We have used gel mobility shift assays to detect proteins that bind to DNA treated in vitro with duocarmycin SA and identified a protein, named duocarmycin-DNA adduct recognizing protein (DARP), which binds with increased affinity to duocarmycin-damaged DNA. Examination with partially purified DARP revealed that the protein recognized not only the DNA adduct of structurally related drug, CC-1065, but unexpectedly, the protein also recognized the DNA adduct of another chemotype of minor groove binder, anthramycin. These results demonstrate that DARP recognizes the structural alteration of DNA induced by these potent DNA-alkylating drugs, suggesting the possibility that the protein might modulate the antitumor activity of these drugs.

Tight association of loss of merlin expression with loss of heterozygosity at chromosome 22q in sporadic meningiomas.

Mutations of NF2, the gene for neurofibromatosis 2, are detected in 20-30% of sporadic meningiomas, and almost all mutations lead to loss of merlin expression. However, loss of heterozygosity (LOH) at chromosome 22q is found at a much higher frequency, up to 50-70%, and the possibility of another tumor suppressor gene in this region has not been excluded. Furthermore, a recent report proposed that abnormal activation of a protease micro-calpain can be an alternative pathway for merlin loss in meningiomas and schwannomas. To determine the correlation of merlin loss with NF2 genetic alteration or micro-calpain activation, we performed a molecular genetic analysis of 50 sporadic meningiomas and also examined the expression status of merlin and active form micro-calpain. LOH assay of five microsatellite markers franking NF2 revealed LOH in 22 cases, and single-strand conformation polymorphism assay detected six frameshift mutations, two splicing mutations, one nonsense mutation, and one missense mutation, all accompanied by 22q LOH. In addition, a multiplex PCR assay indicated homozygous deletion of NF2 in two cases. Interestingly, a marked decrease of merlin expression was seen exclusively in the 22 cases with 22q LOH. Activated micro-calpain expression was observed in 28 cases at various levels but showed no correlation with merlin status. These data strongly support the notion that NF2 is the sole target of 22q LOH in meningiomas and that loss of merlin expression is always caused by genetic alteration of NF2, following the classic "two hit" theory.

Expression level of Bcl-2 determines anti- or proapoptotic function.

Bcl-2 is an oncogene with antiapoptotic function. However, Bcl-2 is converted to a Bax-like death effector by caspases, suggesting that the expression of Bcl-2 may not favor the growth of cancers. We introduced the Bcl-2 gene to gliomas via adenovirus (Adv; Adv-Bcl-2) with the Adv for Fas (Adv-Fas) and the Adv for Fas ligand (Adv-FL) to evaluate the antiapoptotic function of Bcl-2. In U251 glioblastoma cells, Bcl-2 at a low level of expression repressed apoptosis induced by Adv-Fas and Adv-FL, whereas Bcl-2 at a high level of expression did not. On the other hand, Bcl-X(L) showed antiapoptotic function against Fas-mediated apoptosis, irrespective of its expression level. In glioblastoma cells, induction of Bcl-2 alone at a high level induced apoptosis, whereas induction of Bcl-X(L) alone did not. As the multiplicity of infection of Adv-Bcl-2 was increased, the quantity of a cleaved product of Bcl-2 increased. Induction of caspase-inhibitory genes (CrmA and p35) inhibited apoptosis induced by Adv-Bcl-2. Induction of Bcl-2 led to alteration of the membrane potential and structure of the mitochondria. In summary, although Bcl-2 at a low level of expression was antiapoptotic, Bcl-2 at a high level of expression was proapoptotic to Fas-mediated apoptosis. Overexpression of Bcl-X(L) was consistently antiapoptotic to Fas-mediated apoptosis.

Highly augmented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of gliomas.

An E1B 55-kDa gene-defective adenovirus (Adv), ONYX-015, has been reported to be a highly useful replication-competent Adv that shows cytopathic effect for cancers with an abnormal p53 gene, without damaging normal tissues. In this study, we combined this Adv (Adv-E1AdB) with a fiber mutation, F/K20, which has a stretch of 20 lysine residues added at the COOH-terminus of the fiber and shows high transduction efficiency to gliomas. In U-373 MG glioma cells, the transduction efficiency of Adv-F/ K20 for lacZ was nine times higher than that of the Adv with wild-type fiber (Adv-F/wt) for lacZ. At a multiplicity of infection of 30, the replication efficiency of Adv-E1AdB-F/K20 was 11 times higher than that of Adv-E1AdB with wt fiber (Adv-E1AdB-F/wt). The ED50 value of AdvE1AdB-F/K20 to U-373 MG cells, which is a measure of the in vitro cytopathic effect, was 32 times greater than that of Adv-E1AdB-F/wt. injection of Adv-E1AdB-F/K20 suppressed the in vivo growth of tumors. The antitumoral effect of Adv-E1AdB-F/K20 was remarkably stronger than that of Adv-E1AdB-F/wt. A greater quantity of replicated virus protein (hexon) by infection with Adv-E1AdB-F/K20 was demonstrated in vitro and in vivo, compared with that of Adv-E1AdB-F/wt. In conclusion, gene therapy using Adv-E1AdB-F/K20, which drastically augmented the antitumoral effect of Adv-E1AdB, will be a promising therapeutic approach for gliomas.

Characteristics of antitumor activity of KW-2189, a novel water-soluble derivative of duocarmycin, against murine and human tumors.

Methyl(1S)-1-bromomethyl-7-methyl-5-[(4-methylpiperazinyl)-carb onyloxy]- 3-[(5,6,7-trimethoxy-2-indolyl)-carbonyl]-1,2-dihydro-3H-pyrolo[3, 2-e] indole-8-carboxylate hydrobromide (KW-2189), a novel derivative of duocarmycin B2, was selected for extensive evaluation based on its improved antitumor activity, water solubility, and stability in the culture medium, as compared with duocarmycin B2. Although the in vitro cell growth-inhibitory activity of KW-2189 was less potent than that of duocarmycin B2, it significantly inhibited the growth of five murine solid tumors including Colon 26 adenocarcinoma, Colon 38 adenocarcinoma, and B16 melanoma in vivo. KW-2189 was also effective against murine P388 leukemia and L1210 leukemia not only by local administration (i.p.-i.p. system), but also by systemic administration (i.p.-i.v. or i.v.-i.v. system). The most remarkable feature of KW-2189 was its efficacy against various human xenografts, which was observed in 14 tumors among 16 tested tumors including drug-insensitive tumors by single i.v. administration. Tumor regression was observed in mice bearing LC-6 lung, St-4 and St-40 stomach, Li-7 liver, PAN-2 pancreas, and MX-1 breast carcinomas. In many cases, the activities of KW-2189 were more than those of clinically active agents, mitomycin C, Adriamycin, cisplatin, and cyclophosphamide. Delayed lethal toxicity, which was reported in mice treated with CC-1065 whose structure was similar to KW-2189, was not observed in mice treated with KW-2189. KW-2189 inhibited DNA synthesis more significantly than RNA or protein synthesis, although DNA strand breaks were not observed. KW-2189 was activated by porcine liver esterase, mouse liver homogenate or Hep G2 homogenate, and DU-86-DNA adducts were detected in KW-2189-treated HeLa S3 cells, suggesting that KW-2189 was converted to DU-86 in the cells. These results indicate that KW-2189 is an interesting candidate for further development as a novel antitumor agent.

Adenovirus-mediated transfer of p33ING1 with p53 drastically augments apoptosis in gliomas.

The p53 tumor suppressor gene is an important target for the gene therapy of cancers, and clinical trials targeting this gene have been conducted. Some cancers, however, are refractory to p53 gene therapy. Therefore, it has been combined with other therapies, including chemotherapy and radiotherapy, to enhance the cytopathic effect of p53 induction. The p33ING1 gene cooperates with p53 to block cell proliferation. In this study, we investigated whether adenovirus (Adv)-mediated coinduction of p33ING1 and p53 enhances apoptosis in glioma cells (U251 and U-373 MG), which showed no genetic alterations but low expression levels of p33ING1. Although the single infection of Adv for p33ING1 (Adv-p33) at a multiplicity of infection (MOI) of 100, or Adv for p53 controlled by myelin basic protein (MBP) promoter (Adv-MBP-p53), a glioma-specific promoter, at a MOI of 50, did not induce apoptosis in U251 and U-373 MG glioma cells; coinfection of Adv-p33 and Adv-MBP-p53 at the same MOIs induced drastically enhanced apoptosis in both cell lines. Apoptosis was not induced in NGF-treated PC-12 cells infected with a high MOI (300) of Adv-p33 nor in those coinfected with Adv-p33 (100) and Adv-MBP-p53 (50). Coinfection of Adv-p33 and Adv-MBP-p53 demonstrated morphological mitochondrial damage during the initial stage of apoptosis, which likely led to apoptotic cell death. Our results indicate that this coinfection approach can be used as a modality for the gene therapy of gliomas, sparing damage to normal tissues.

Risk Factors for the Discontinuation of Home Medical Care among Low-functioning Older Patients.

OBJECTIVES: Older patients receiving home medical care often have declining functional status and multiple disease conditions. It is important to identify the risk factors for care transition events in this population in order to avoid preventable transitions. In the present study, therefore, we investigated the factors associated with discontinuation of home medical care as a potentially preventable care transition event in older patients. METHODS: Baseline data for participants in the Observational study of Nagoya Elderly with HOme MEdical (ONEHOME) study and data on the mortality, institutionalization, or hospitalisation of the study participants during a 2-year follow-up period were used. Discontinuation of home care was defined as admission to a hospital for any reason, institutionalization, or death. Univariate and multivariate Cox hazard models were used to assess the association of each of the factors with the discontinuation of home care during the observational period. The covariates included in the multivariate analysis were those significantly associated with the discontinuation of home care at the level of P<0.05 in the univariate analysis. RESULTS: The univariate Cox hazard model revealed that a low hemoglobin level (< 11g/dL), low serum albumin level (< 3g/dL), higher Charlson Comorbidity Index score, and low Mini Nutritional Assessment Short Form score (< 7) were significantly associated with the discontinuation of home care. A multivariate Cox hazard model including these four factors demonstrated that all four were independently associated with home-care discontinuation. CONCLUSIONS: The present results demonstrated that anemia, hypoalbuminemia, malnourishment, and the presence of serious comorbidities were associated with the discontinuation of home medical care among low-functioning older patients.

Relative level of expression of Bax and Bcl-XL determines the cellular fate of apoptosis/necrosis induced by the overexpression of Bax.

The Bax protein plays a critical role in the apoptosis of cancers induced by radiotherapy or chemotherapy, which induce both apoptosis and necrosis. We transduced various glioblastoma cells with the Bax gene via an adenoviral vector and found that A-172 cells led to necrotic cell death, while U251 cells apoptotic cell death, even though a similar level of Bax protein was introduced. A-172 cells displayed a much higher constitutive expression of the Bcl-XL protein compared with that of U251 cells. Upon simultaneous overexpression of the Bcl-XL and Bax proteins in the U251 cells, Bax-induced apoptosis of U251 cells was suppressed and an increase in the number of necrotic cells was seen. Moreover, induction of a higher amount of Bax protein in A-172 cells increased the percentage of apoptotic cells. In conclusion, if a cancerous cell expresses a high enough amount of Bax to undergo death, apoptosis will be induced. If a cancerous cell expresses a level of Bcl-XL which prevents Bax-induced apoptosis, the overexpression of Bax leads to necrotic cell death.

The s-Myc protein having the ability to induce apoptosis is selectively expressed in rat embryo chondrocytes.

Gene transfection experiments demonstrated that over-expression of the s-myc gene under the control of a human metallothionein promoter induced apoptosis in cells such as rat and human glioma cells. In contrast to c-Myc-mediated apoptosis requiring withdrawal of serum growth factors, s-myc expression induced apoptosis in glioma cells in the presence of 10% fetal calf serum. Whereas, s-Myc-mediated apoptosis was suppressed in proportion to the increase of bcl-2 expression as seen in c-Myc mediated apoptosis. The s-myc gene was expressed in rat embryo cells being committed to differentiate to hypertrophic chondrocytes which undergo programmed cell death. CAT assay demonstrated that in the NH2-terminal region, the s-Myc protein contains a domain structure required for expression of transactivation activity that is approximately six times higher than that of c-Myc. Therefore, these findings strongly suggest that s-Myc may play an important role in transcription regulation of a set of genes whose expression induces programmed cell death in vitro and in vivo.

Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation.

Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that, similar to Bcl-2, Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, we examined the effect of Pim-1 expression on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Our results showed that, rather than inhibiting apoptosis, Pim-1 expression stimulated c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulated c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolished the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant failed to stimulate c-Myc-mediated apoptosis, and Pim-1 expression alone in the absence of c-Myc overexpression did not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway were required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. Our results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) which regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation.

Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis.

Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. In this study, we found that the overexpression of the Bcl-2 family member Bax induced apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleaved the DEVD tetrapeptide. This apoptotic cell death was suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. We also found that the Bax-induced apoptosis of COS-7 cells was suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevented etoposide-induced apoptosis in COS-7 cells. In addition, Bcl-xL inhibited the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death but Bcl-2 did not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells could be differentially regulated. Our results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity.

Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells.

To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated 'type 2 physiological cell death' or 'autophagic degeneration', and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.

Proteasome inhibitors induce cytochrome c-caspase-3-like protease-mediated apoptosis in cultured cortical neurons.

The ubiquitin-proteasome protein degradation pathway is crucial in controlling intracellular levels of a variety of short-lived proteins and maintaining cellular growth and metabolism. In a previous study, we showed the accumulation of conjugated ubiquitin in CA1 neurons of the gerbil after 5 min of forebrain ischemia (; ). The accumulation of conjugated ubiquitin may reflect proteasome malfunction. In the present study, we investigated the effects of proteasome inhibitors on primary neuronal cultures to determine whether proteasomal malfunction induces neuronal death. When carbobenzoxy-Leu-Leu-Leu-aldehyde or lactacystin, two different types of proteasome inhibitors, were separately used to suppress proteasome activity, we observed induction of apoptotic neuronal cell death in both cases. During the apoptotic process, mitochondrial membrane potential was disrupted, cytochrome-c was released from mitochondria into the cytosol, and caspase-3-like proteases were activated. Apoptosis was inhibited by pretreatment with acetyl-aspartyl-glutamyl-valyl-aspart-1-aldehyde or overexpression of Bcl-x/(L). These results demonstrated that suppression of proteasome function induces neuronal apoptosis via the release of cytochrome c from mitochondria and activation of caspase-3-like proteases.

s-Myc acts as a transcriptional activator and its sequence-specific DNA binding is required for induction of programmed cell death in glioma cells.

We previously reported that s-Myc expression effectively induces programmed cell death (PCD) by apoptosis in glioma cells that express only mutated p53. To determine the molecular mechanism of s-Myc-induced PCD, we examined the correlation between transcriptional activation of s-Myc and its ability to induce PCD. Using a reporter plasmid having an upstream promoter region containing four repeats of the hexanucleotide CACGTG, we found that s-Myc can activate transcription of a reporter gene from this plasmid. Two mutated forms of s-Myc protein, s-MycCKII and s-MycmBR, were created. While s-MycCKII whose casein kinase (CK) II cognate sequence was restored in the internal acidic domain activated transcription as efficiently as wild-type s-Myc and induced PCD in glioma cells, s-MycmBR having a mutated basic region unable to bind the CACGTG motif did not. These findings suggest that transactivation activity of s-Myc through sequence-specific DNA binding may be indispensable for induction of PCD but that lack of a CK-II cognate sequence in the internal acidic domain may have little effect on these functions of s-Myc.

Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas: implication for gene therapy.

Caspase-8 is a member of the family of caspases, which are involved in the execution of apoptosis. To investigate whether caspase-8 can be used for gene therapy of gliomas, we transduced A-172 and U251 glioma cells with the caspase-8 gene via an adenoviral vector (Adv) controlled by the chicken beta-actin (CA) promoter (Advcaspase-8), and found that a similar level of caspase-8 protein induced A-172 cells to undergo necrotic cell death and U251 cells to undergo apoptotic cell death. Neither Bcl-XL nor Bcl-2, which play important roles in antiapoptotic mechanisms in gliomas, protected glioma cells from apoptosis induced by overexpression of caspase-8. Injection of Adv-caspase-8 suppressed the in vivo growth of U251 xenografts, in which apoptotic cell death remarkably increased as revealed by TUNEL analysis. Finally, we assessed whether gene therapy with a tissue-specific promoter, the myelin basic protein (MBP) promoter, is applicable to gliomas. Adv for caspase-8 controlled by the MBP promoter induced drastic apoptosis in U251 and U-373MG glioma cells, whereas it did not induce apoptosis in human endothelial cells, fibroblasts, and nerve growth factor-treated PC12 cells. These results indicate that Adv for caspase-8 effectively induced cell death in gliomas, and that this approach may be a useful modality for gene therapy of gliomas.

Construction, propagation, and titer estimation of recombinant adenoviruses carrying proapoptotic genes.

Generation of a recombinant adenovirus (Adv) that induces the constitutive expression of an apoptotic gene has been extremely difficult owing to severe apoptotic damage to the host cell. In this study, 293 cells were transduced with the caspase-inhibiting CrmA gene (293-CrmA cells), and used as host cells to generate Adv carrying apoptosis-inducing genes (proapoptotic genes). The 293-CrmA cells proved to be highly efficient for the construction of recombinant Adv carrying genes encoding Fas and Fas ligand. Moreover, the 293-CrmA line produced an ample quantity of these recombinant viruses. Because the conventional 293 plaque formation assay did not reflect the actual number of cells infected with the Adv carrying the proapoptotic gene, a determination of the Adv DNA copy number introduced into target cells was necessary to evaluate the quantity of infective virus. The techniques described here should be widely applicable for the construction of a recombinant Adv, in ample quantity, and for the estimation of the quantity of recombinant Adv produced.