Takao Kanzawa

Pivotal Role of the Cell Death Factor BNIP3 in Ceramide-Induced Autophagic Cell Death in Malignant Glioma Cells

The sphingolipid ceramide has been recognized as an important second messenger implicated in regulating diverse signaling pathways especially for apoptosis. Very little is known, however, about the molecular mechanisms underlying nonapoptotic cell death induced by ceramide. In the present study, we first demonstrate that ceramide induces nonapoptotic cell death in malignant glioma cells. The cell death was accompanied by several specific features characteristic of autophagy: presence of numerous autophagic vacuoles in the cytoplasm, development of the acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3), and a marked increase in expression levels of two forms of LC3 protein (LC3-I and LC3-II). We additionally demonstrate that ceramide decreases mitochondrial membrane potential and activates the transcription of death-inducing mitochondrial protein, BNIP3, resulting in increased expression levels of its mRNA and protein in malignant glioma cells. Moreover, tumor cells transfected with BNIP3 gene undergo autophagy in the absence of ceramide. These results suggest that ceramide induces autophagic cell death in malignant glioma cells via activation of BNIP3. This study adds a new concept to characterize the pathways by which ceramide acts to induce nonapoptotic autophagic cell death in malignant gliomas.

Telomerase as a therapeutic target for malignant gliomas

Telomerase, a ribonucleoprotein enzyme, is considered as a potential target of cancer therapy because of its preferential expression in tumors. In particular, malignant gliomas are one of the best candidates for telomerase-targeted therapy. It is because malignant gliomas are predominantly telomerase-positive, while normal brain tissues do not express telomerase. In theory, there are two telomerase-associated therapeutic approaches for telomerase-positive tumors. One approach is the anti-telomerase cancer therapy to directly inhibit telomerase activity, resulting in apoptotic cell death or growth arrest. Two major components of the telomerase holoenzyme complex, the RNA template (hTER) and catalytic subunit (reverse transcriptase, hTERT) are well considered as therapeutic targets. The other approach is the telomerase-specific cancer therapy by targeting telomerase-expressing tumor cells as a means to directly kill the cells. Strategies using the transfer of therapeutic gene under the hTERT promoter system as well as immunotherapy directed against telomerase-positive cells are generally included. These telomerase-associated therapies are very promising for the treatment of malignant gliomas.

Mild Heat Shock Induces Autophagic Growth Arrest, But Not Apoptosis in U251-MG and U87-MG Human Malignant Glioma Cells

Although hyperthermia has been used as a treatment of malignant brain tumors, it is not yet clear what is the mechanism of the cell growth inhibition by heat shock, especially by the temperature which has clinically been applied to tumor–brain border-zone, 42–43°C. Therefore, we evaluated the change of U251-MG and U87-MG human malignant glioma cells after 43°C-heat shock comparing with that of 45°C. First, we observed that cell growth was transiently inhibited after 43°C-heat shock for 3 or 5 days, in U251-MG or U87-MG cells, respectively, which was followed by regrowth. During the period of transient growth inhibition, mild G2/M arrest was observed. However, apoptosis was observed in only 2.7% or 1.5%, of 43°C-heated cells, in U251-MG or U87-MG cells, respectively. Instead, transmission electron micrography showed the formation of vacuoles, degeneration of mitochondria, and autophagosomes. Moreover, in the both cell lines, flow-cytometric analysis with acridine orange revealed the induction of acidic vesicle organelles, which was blocked by 3-methyladenine (3-MA), suggesting the involvement of autophagy. Furthermore, while 3-MA did not increase the anti-tumor effect of 43°C-heat shock, bafilomycin A1, another autophagy inhibitor, did significantly enhance the effect in U251-MG cells. Taken together, mild heat shock (43°C for 2 h) causes autophagy and mild G2/M arrest, but does not induce apparent apoptosis in U251-MG and U87-MG glioma cells. Inhibition of autophagy with bafilomycin A1 may increase the anti-tumor efficacy of mild heat shock against some malignant glioma cells.

Caspase-8 Gene Therapy Using the Human Telomerase Reverse Transcriptase Promoter for Malignant Glioma Cells

Telomerase is a distinctive candidate for targeted gene therapy of malignant gliomas, because the vast majority of malignant gliomas express telomerase activity while normal brain tissues do not. Recently, we developed a telomerase-specific expression system of caspase-8 gene using the promoter of the human telomerase reverse transcriptase (hTERT) gene. However, the transcriptional activity of hTERT-181 promoter (a 181-base pair [bp] region upstream of the transcription start site) was relatively lower in malignant glioma cells than in other tumors such as prostate cancer cells. To establish the hTERT/caspase-8 construct as a novel therapy for malignant gliomas, we need to increase the transcriptional activity of the hTERT promoter in malignant glioma cells. In the present study, we demonstrate that the transcriptional activity of hTERT-378 promoter (a 378-bp region) was 2- to 40-fold higher in hTERT-positive malignant glioma cells (A172, GB-1, T98G, U87-MG, U251-MG, and U373-MG) than that of hTERT-181. We further demonstrate that by using the hTERT-378/caspase-8 construct, apoptosis was restricted to malignant glioma cells, and was not seen in astrocytes or fibroblasts lacking hTERT. Moreover, the growth of subcutaneously established U373-MG tumors in mice was significantly inhibited by seven daily intratumoral injections of hTERT-378/caspase-8 construct and its inhibitory effect persisted during 3 additional weeks without additional treatment. These results suggest that the telomerase-specific expression of caspase-8 under hTERT-378 promoter is a novel targeting approach for the treatment of telomerase-positive malignant gliomas.

Differentiated regulation of allo‐antigen presentation by different types of murine microglial cell lines

We established granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐dependent murine microglial clones and investigated the immune properties of four individual clones. All four clones expressed MHC class I and CD54 (ICAM‐1) at similar levels. The 5‐2, Ra2, and 6‐3 clones expressed CD80 (B7‐1), CD86 (B7‐2), and MHC class II at low, medium, and high levels, respectively. Only the 6‐3 clone expressed CD40. Generally, the levels of co‐stimulation and CD 40 signals had a profound effect on the response to antigens. The 5‐2, Ra2, and 6‐3 clones, however, stimulated allogenic T‐cell proliferation to the same extent or less compared to spleen cells. Although the 6‐1 clone expressed co‐stimulatory and MHC molecules at levels similar to Ra2, it suppressed allogenic T‐cell proliferation, unlike Ra2. Thus, allo‐antigen presentation by microglial clones was not correlated with the expression of CD40 and co‐stimulatory molecules. When microglial clones were fixed with paraformaldehyde, they enhanced IL‐2‐dependent T‐cell proliferation according to the level of their expression of co‐stimulatory molecules. Furthermore, conditioned medium from the 6‐1 clone inhibited the T‐cell response to allo‐antigen. This indicates that some factor(s) derived from a microglial subtype may play an important role in the regulation of T‐cell proliferation in addition to the molecules involved in antigen presentation. Moreover, these results also suggest that there may be specialized subtypes of microglia that regulate the immune response in the CNS. J. Neurosci. Res. 62:383–388, 2000.

Current and Future Gene Therapy for Malignant Gliomas.

Malignant gliomas are the most common neoplasm in the central nervous system. When treated with conventional treatments including surgery, irradiation, and chemotherapy, the average life expectancy of the most malignant type, glioblastoma multiforme is usually less than 1 year. Therefore, gene therapy is expected to be an effective and possibly curative treatment. Many gene therapeutic approaches have demonstrated efficacy in experimental animal models. However, the current clinical trials are disappointing. This review focuses on current therapeutic genes/vectors/delivery systems/targeting strategies in order to introduce updated trends and hopefully indicate prospective gene therapy for malignant gliomas.