Tadashi Komata

A novel telomerase-specific gene therapy : gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter

Apoptosis is a genetically encoded cell death process and is a pathway that may be disrupted in tumor cells. Therefore, therapies that restore the ability to undergo apoptosis are promising for the treatment of tumor cells. We have demonstrated that the transfer of apoptosis-inducible genes inhibits the growth of tumors in vitro and in vivo through induction of apoptosis. However, to restrict induction of apoptosis to tumor cells, we need to explore a tumor-specific expression system of these genes. In the present study, we developed the telomerase-specific transfer system of apoptosis-inducible genes, utilizing the promoter of the human telomerase catalytic subunit (hTERT) gene. Approximately 90% of tumors have telomerase activity whereas most normal cells do not express the activity. These observations indicate that telomerase is a particularly attractive target for the tumor-specific expression system of vectors. We demonstrate here that by using the hTERT promoter-driven caspase-8 expression vector (hTERT/caspase-8), apoptosis is restricted to telomerase-positive tumor cells of wide range, and is not seen in normal fibroblast cells without telomerase activity. Furthermore, treatment of subcutaneous tumors in nude mice with the hTERT/caspase-8 construct inhibited tumor growth significantly because of induction of apoptosis (p < 0.01). The telomerase-specific expression of apoptosis-inducible genes afforded by the hTERT promoter, therefore, may be a novel and promising targeting approach for the treatment of tumors with telomerase activity.

Treatment of prostate cancer in vitro and in vivo with 2-5A-anti-telomerase RNA component

Prostate cancer is the most common malignancy of elderly men in the United States. Since there is no curative treatment for advanced prostate cancer, exploration of novel modalities of treatment is essential. Telomerase, a ribonucleoprotein, is detected in the vast majority of prostate cancer, but not in normal or benign prostatic hyperplasia tissues. Thus, telomerase is expected to be a very strong candidate for targeted therapy of prostate cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and examined its cytotoxic effect on prostate cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. We here show that treatment with 2-5A-anti-hTR in the presence of a cationic liposome reduced cell viability of tumor cell lines tested to 9–18% within 6 days. In contrast, normal fibroblast cells were resistant to the treatment. Its effect was mainly due to induction of apoptosis by activated caspase family members. Furthermore, treatment of subcutaneous tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P<0.001). The treatment with 2-5A-anti-hTR may be a promising strategy for the treatment modality of prostate cancer with telomerase activity.

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.

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.

B7-1(CD80)-transfected human glioma cells and interleukin-12 directly stimulate allogeneic CD8+ T cells.

To obtain more effective cytotoxic T lymphocytes (CTLs), we examined the effect of B7 costimulation and interleukin-12 (IL-12) on the induction of allogeneic CTLs. Peripheral blood lymphocytes (PBLs) or positively selected human CD8+ T cells (purity > 99%) from healthy donors were used as effector cells, and B7-1-transfected or nontransfected U251 human glioma cells (U251-B7 or U251-Vec.) were used as stimulator cells. In mixed lymphocyte culture (MLC) with PBLs and U251 cells, nonspecific natural killer (NK) activity was raised by addition of IL-12, and the effect of B7 costimulation was not observed. However, in MLC with CD8+ T cells, efficient proliferation, generation of CTLs, and cytokine production were induced by MLC with U251-B7 in the presence of IL-12. Efficient generation of CTLs was not induced by either MLC with U251-B7 alone or the addition of IL-12 alone. Our results indicate that a combination of B7-1 costimulation and IL-12 is effective for inducing generation of CTLs, and that the CD8+ T cells can be differentiated into CTLs without the help of CD4+ T cells or antigen-presenting cells or both.