Faculty Profiles - FUJIWARA Toshiyoshi

Visualization of intrathoracically disseminated solid tumors in mice with optical imaging by telomerase-specific amplification of a transferred green fluorescent protein gene

Tatsuo Umeoka, Takeshi Kawashima, Shunsuke Kagawa, Fuminori Teraishi, Masaki Taki, Masahiko Nishizaki, Satoru Kyo, Katsuyuki Nagai, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara

Cancer Research 64 ( 17 ) 6259 - 6265 2004.9

Language：English

Currently available methods for detection of tumors in vivo such as X-ray, computed tomography, and ultrasonography are noninvasive and have been well studied
the images, however, are not specific for tumors. Direct optical imaging of tumor cells in vivo that can clearly distinguish them from surrounding normal tissues may be clinically useful. Here, we describe a new approach to visualizing tumors whose fluorescence can be detected using tumor-specific replication-competent adenovirus (OBP-301, Telomelysin) in combination with Ad-GFP, a replication-deficient adenovirus expressing green fluorescent protein (GFP). Human telomerase reverse transcriptase is the catalytic subunit of telomerase, which is highly active in cancer cells but quiescent in most normal somatic cells. We constructed an adenovirus 5 vector in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site and showed that OBP-301 replicated efficiently in human cancer cells, but not in normal cells such as human fibroblasts. When the human lung and colon cancer cell lines were infected with Ad-GFP at a low multiplicity of infection, GFP expression could not be detected under a fluorescence microscope
in the presence of OBP-301, however, Ad-GFP replicated in these tumor cells and showed strong green signals. In contrast, coinfection with OBP-301 and Ad-GFP did not show any signals in normal cells such as fibroblasts and vascular endothelial cells. We also found that established subcutaneous tumors could be visualized after intratumoral injection of OBP-301 and Ad-GFP. A549 human lung tumors and SW620 human colon tumors transplanted into BALB/c nu/nu mice were intratumorally injected with 8 × 105 plaque-forming units of Ad-GFP in combination with 8 × 106 plaque-forming units of OBP-301. Within 3 days of treatment, the fluorescence of the expressed GFP became visible by a three-chip color cooled charged-coupled device camera in these tumors, whereas intratumoral injection of Ad-GFP alone could not induce GFP fluorescence. Moreover, intrathoracic administration of Ad-GFP and OBP-301 could visualize disseminated A549 tumor nodules in mice after intrathoracic implantation. Our results indicate that intratumoral or intrathoracic injection of Ad-GFP in combination with OBP-301 might be a useful diagnostic method that provides a foundation for future clinical application.

DOI： 10.1158/0008-5472.CAN-04-1335

テロメラーゼ特異的制限増殖型アデノウイルスTelomelysinと微小管作用型抗癌剤の併用効果の検討

藤原俊哉, 藤原俊義, 香川俊輔, 滝正樹, 岸本浩行, 渡辺貴紀, 遠藤芳克, 京哲, 水口裕之

日本癌学会総会記事 63rd 510 2004.8

PPS-2-026 p53蛋白質をパルスしたヒト抹消血単核球由来の樹状細胞を用いた胃癌の新しい免疫療法(胃分子生物2)

香川俊輔, 藤原俊義, 徳永尚之, 西崎正彦, 松岡順治, 合地明, 田中紀章

日本消化器外科学会雑誌 37 ( 7 ) 1214 - 1214 2004.7

Antitumor effect of intratumoral administration of bone marrow-derived dendritic cells transduced with wild-type p53 gene

Takayoshi Murakami, Naoyuki Tokunaga, Toshihiko Waku, Shinya Gomi, Shunsuke Kagawa, Noriaki Tanaka, Toshiyoshi Fujiwara

Clinical Cancer Research 10 ( 11 ) 3871 - 3880 2004.6

Language：English

Purpose: Dendritic cells (DCs) are attractive effectors for cancer immunotherapy because of their potential to function as professional antigen-presenting cells for initiating cellular immune responses. The tumor suppressor gene p53 is pivotal in the regulation of apoptosis, and ∼50% of human malignancies exhibit mutation and aberrant expression of p53. We investigated the antitumor effect of intratumoral administration of bone marrow-derived dendritic cells transduced with wild-type p53 gene. Experimental Design: We examined whether intratumoral administration of DCs infected with recombinant adenovirus expressing murine wild-type p53 (Ad-mp53) could induce systemic antitumor responses against mutant p53-expressing tumors, highly immunogenic MethA, or weakly immunogenic MCA-207 implanted in syngeneic mice. Results: Accumulation of wild-type p53 protein in bone marrow-derived murine DCs could he successfully achieved by Ad-mp53 infection. Treatment with intratumoral injection of Ad-mp53-transduced DCs caused a marked reduction in the in vivo growth of established MethA and MCA-207 tumors with massive cellular infiltrates. Administration of p53-expressing DCs suppressed the growth of both injected MCA-207 tumors and untreated distant MCA-207 tumors, but not unrelated Lewis lung carcinoma tumors, suggesting the augmentation of systemic immunogenicity against MCA-207 tumor cells. Moreover, intratumoral injection of p53-expressing DCs had a greater antitumor effect than did s.c. immunization. Conclusions: Our results indicate that intratumoral administration of DCs expressing murine wild-type p53 leads to significant systemic immune responses and potent antitumor effects in mutant p53-expressing murine cancer models. These findings raise the possibility of using this strategy of intratumoral injection of p53-expressing DCs for human cancer treatment.

DOI： 10.1158/1078-0432.CCR-03-0599

Thoracic tumors treated with CT-guided radiofrequency ablation: Initial experience

K Yasui, S Kanazawa, Y Sano, T Fujiwara, S Kagawa, H Mimura, S Dendo, T Mukai, H Fujiwara, T Iguchi, T Hyodo, N Shimizu, N Tanaka, Y Hiraki

RADIOLOGY 231 ( 3 ) 850 - 857 2004.6

Language：English Publisher：RADIOLOGICAL SOC NORTH AMERICA

PURPOSE: To determine the effectiveness of computed tomography (CT)-guided radiofrequency (RF) ablation of malignant thoracic tumors.
MATERIALS AND METHODS: CT-guided RF ablations of 99 malignant thoracic tumors (3-80 mm in largest diameter; mean, 19.5 mm) were performed in 35 patients in 54 sessions. Ablation was performed with an RF generator by using a single internally cooled electrode. Tumors were both primary (three lesions) and, secondary (pulmonary or pleural metastases, 96 lesions). Follow-up was 1-17 months (mean, 7.1 months). Follow-up CT and histopathologic examinations were evaluated. Univariate analysis was performed with the Fisher exact test and Welch, t test was used to evaluate differences between group means. P < .05 represented a significant difference. The maximal diameter of each residual tumor or local recurrence or the proportion of primary lesions of pulmonary metastatic tumors with recurrence after RF ablation were analyzed. Complications, management, and outcomes of the complications were recorded.
RESULTS: The appearance of each ablation zone, including the target tumor and surrounding normal lung parenchyma, showed involution at follow-up CT. Local recurrence was demonstrated histopathologically or radiologically in nine tumors. The other 90 tumors showed no growth progression at follow-up CT. Probable complete coagulation necrosis obtained with initial RF ablation was achieved in 91% (90 of 99) of the tumors. The mean maximal diameter of the nine tumors (19.6 mm +/- 7.7 [SD]) was not significantly different (P = .994) from that of the other 90 tumors (19.5 mm +/- 13.0). Primary lesions of those nine metastatic tumors varied and did not demonstrate a specific tendency. Complications included pneumothorax, fever higher than 37.5&DEG;C, hemoptysis, cough, pleural effusion, abscess formation, and hemothorax. The overall complication rate was 76% (41 of 54 sessions).
CONCLUSION: RF ablation seems to be a promising treatment for malignant (C) RSNA, 2004.

DOI： 10.1148/radiol.2313030347

Late resistance to adenoviral p53-mediated apoptosis caused by decreased expression of Coxsackie-adenovirus receptors in human lung cancer cells

Yasuhisa Tango, Masaki Taki, Yoshiko Shirakiya, Shoichiro Ohtani, Naoyuki Tokunaga, Yosuke Tsunemitsu, Shunsuke Kagawa, Toru Tani, Noriaki Tanaka, Toshiyoshi Fujiwara

Cancer Science 95 ( 5 ) 459 - 463 2004.5

Language：English

Adenovirus-mediated wild-type p53 gene transfer induces apoptosis in a variety of human cancer cells. Although clinical trials have demonstrated that a replication-deficient recombinant adenovirus expressing the wild-type p53 gene (Ad-p53) is effective in suppressing growth of non-small cell lung cancer (NSCLC), we often experienced late resistance to this treatment. To elucidate the mechanism of late resistance to Ad-p53 in human lung cancer cells, we generated 5 different resistant variants from p53-susceptible H1299 NSCLC cells by repeated infections with Ad-p53. We first examined the transduction efficiency of adenoviral vector by Ad-LacZ transduction followed by X-gal staining in parental and 5 resistant H1299 cell lines. Their sensitivity to viral infection decreased in correlation with the magnitude of resistance, and Ad-p53-mediated tumor suppression could be restored by dose escalation of Ad-p53 in the resistant variants. The expression of Coxsackie and adenovirus receptor (CAR) and αV integrins, which are cellular receptors for attachment and internalization of the virus, respectively, was next investigated in these cell lines. Flow cytometry revealed that αVβ3 and αVβ5 integrin expression was consistent, while p53-resistant cell lines showed that diminished CAR expression correlated with the magnitude of the resistance. Our results demonstrated that decreased CAR expression could be one of the mechanisms of late resistance to Ad-p53, which may have a significant impact on the outcome of adenovirus-based cancer gene therapy.

DOI： 10.1111/j.1349-7006.2004.tb03232.x

Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer

Y Tsunemitsu, S Kagawa, N Tokunaga, S Otani, T Umeoka, JA Roth, B Fang, N Tanaka, T Fujiwara

GUT 53 ( 4 ) 554 - 560 2004.4

Language：English Publisher：B M J PUBLISHING GROUP

Background: Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/ PGK-GV16+ Ad/ GT-Bax: Ad/Bax).
Aims: To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53.
Methods: The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/ Bax or Ad/ p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/ Bax and Ad/ p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment.
Results: Treatment with Ad/ Bax and Ad/ p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/ p53 and only treatment with Ad/ Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/ Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/ p53 treatment.
Conclusion: Ad/ Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.

DOI： 10.1136/gut.2003.021683

制限増殖型ウイルス製剤を用いたMolecular Surgery:癌微小転移に対する治療戦略として

滝正樹, 藤原俊義, 水口裕之, 岸本浩行, 藤原俊哉, 香川俊輔, 西崎正彦, 京哲, 田中紀章

日本外科学会雑誌 105 278 2004.3

PS-156-3 非小細胞肺癌に対するp53を標的とした新たな遺伝子治療の前臨床試験 : 化学療法/ 放射線療法/ p53遺伝子治療の3者併用療法の基礎実験

西崎正彦, 藤原俊義, 香川俊輔, Roth Jack A., 田中紀章

日本外科学会雑誌 105 580 - 580 2004.3

Preoperative concurrent chemoradiotherapy with cisplatin and docetaxel in patients with locally advanced non-small-cell lung cancer

H Katayama, H Ueoka, K Kiura, M Tabata, T Kozuki, M Tanimoto, T Fujiwara, N Tanaka, H Date, M Aoe, N Shimizu, M Takemoto, Y Hiraki

BRITISH JOURNAL OF CANCER 90 ( 5 ) 979 - 984 2004.3

Language：English Publisher：NATURE PUBLISHING GROUP

The objective of this study was to assess the feasibility and effectiveness of an induction chemoradiotherapy regimen followed by surgery in patients with locally advanced non-small-cell lung cancer (LA-NSCLC). A total of 22 patients with LA-NSCLC were treated with induction chemoradiotherapy consisting of cisplatin (40 mg m(-2)) and docetaxel (40 mg m(-2)) given on days 1, 8, 29 and 36 plus concurrent thoracic irradiation at a dose of 40-60 Gy (2 Gy fraction(-1) day(-1)). Surgical resection was performed within 6 weeks after completion of induction therapy. Objective response to the induction therapy was obtained in 16 patients (73%). In all, 20 patients (91%) underwent surgery and complete resection was achieved in 19 patients (86%). Pathological downstaging and pathological complete response were obtained in 14 (64%) and five (23%) patients, respectively. With a median follow-up period of 32 months, the calculated 3-year overall and progression-free survival rates were 66 and 61%, respectively. It is noteworthy that the 3-year overall survival rate in 14 patients achieving pathological downstaging was extremely high (93%). Toxicity was manageable with standard approaches. No treatment-related deaths occurred. This combined modality treatment is feasible and highly effective in patients with LA-NSCLC. The results warrant further large-scale study to confirm the effectiveness of this regimen.

DOI： 10.1038/sj.bjc.6601624

Telomerase-Specific Replication-Selective Virotherapy for Human Cancer

Takeshi Kawashima, Shunsuke Kagawa, Naoya Kobayashi, Yoshiko Shirakiya, Tatsuo Umeoka, Fuminori Teraishi, Masaki Taki, Satoru Kyo, Noriaki Tanaka, Toshiyoshi Fujiwara

Clinical Cancer Research 10 ( 1 I ) 285 - 292 2004.1

Language：English

Purpose: Replication-selective tumor-specific viruses present a novel approach for treating neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Telomerase activation is considered to be a critical step in carcinogenesis, and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We investigated the antitumor effect of the hTERT-specific replication-competent adenovirus on human cancer cells. Experimental Design: We constructed an adenovirus 5 vector [tumor- or telomerase-specific replication-competent adenovirus (TRAD)], in which the hTERT promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, and we examined the selective replication and antitumor effect in human cancer cells in vitro and in vivo. Results: TRAD induced selective E1A and E1B expression in human cancer cells, but not in normal cells such as human fibroblasts. TRAD replicated efficiently and induced marked cell killing in a panel of human cancer cell lines, whereas replication as well as cytotoxicity was highly attenuated in normal human fibroblasts lacking telomerase activity. In nu/nu mice carrying s.c. human lung tumor xenografts, intratumoral injection of TRAD resulted in a significant inhibition of tumor growth. No evidence of TRAD was identified in tissues outside of the tumors, despite the presence of TRAD in the circulation. Moreover, TRAD replication in the distant, noninjected tumors was demonstrated. Conclusions: Our results suggest that the hTERT promoter confers competence for selective replication of TRAD in human cancer cells, an outcome that has important implications for the treatment of human cancers.

DOI： 10.1158/1078-0432.CCR-1075-3