2001年５月２日　年報その２

大学では、教授になってもいろいろ提出するものが多くてやっかいです。以下は、札幌医科大学の英語版の年報の日本語訳という複雑な生産物です。これもコピーして使って。

濱田洋文

Department of Molecular Medicine

Our research interests are directed at the elucidation of the molecular mechanisms underlying disease and the application for the better treatment of patients.  The ongoing research fields are: 1) basic and preclinical studies for gene therapy for cancer, immune/inflammatory disorders, and vascular diseases; 2) basic stem cell biology and preclinical studies in regenerative medicine.

難治疾患の根底に潜む分子機構の解明と、その理解に基づいた新しい治療法の開発とを研究の基幹に据えている。 １）癌、免疫・炎症疾患、動脈硬化、虚血性心疾患、などに対する遺伝子治療の基礎ならびに臨床研究、２）幹細胞の生物学と再生医学の前臨床研究、の二つが、研究の柱になっている。

Professor and Chair

Hirofumi Hamada, M.D., Ph.D.

Interests: gene therapy, regenerative medicine, molecular biology

教授、主任

濱田洋文、医学博士

研究領域：　遺伝子治療、再生医学、分子生物学

Instructor

Yoshinori Ito, M.D.

Katsunori Sasaki, Ph.D.

Masayoshi Kobune, M.D., Ph.D.

Sachie Hirai

助手

伊藤克礼

佐々木勝則、医学博士

小船雅義、医学博士

平井幸恵

1. Gene therapy: basic biotechnology

    a) Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma (Yoshida, Y. et al.  Human Gene Therapy  9(17) : 2503-2515, 1998)   Recombinant adenovirus (Adv)-mediated gene transduction is a powerful technology for cancer gene therapy.  In this paper, we report the generation of a fiber-mutant Adv vector, using the Adv genomic DNA-terminal protein complex (DNA-TPC) cotransfection method.  By this procedure, we have generated a fiber mutant, F/K20, which has a linker and a stretch of 20 lysine residues added at the C-terminus of the fiber.  Using Adv for a reporter LacZ gene (AxCAZ2) with either F/K20 or wild-type fiber (F/wt), we examined the transduction efficiency of F/K20-Adv.  The F/K20-Adv showed a remarkably enhanced efficiency in genetic transduction of human glioma cells.  In all four human glioma lines tested, the multiplicities of infection (MOIs) for transduction of 50% of the population (ED₅₀) were decreased with the F/K20-Adv compared with the F/wt-Adv:  by 7-fold for T98G, 14-fold for U251, 9-fold for U373, and 42-fold for U87 cells.  Therefore, we attempted to apply the F/K20-Adv for gene therapy of malignant glioma.  Glioma cells infected with the F/K20-Adv for interleukin-2 or interleukin-12 produced a high level of each cytokine at a much smaller MOI than that with the F/wt-Adv.  Infection with the F/K20-Adv for wild-type p53 tumor suppressor gene resulted in an enhanced level of the p53 protein expression and an increased apoptotic cell death of glioma cells.  These data demonstrated a definite advantage of F/K20-Adv in transduction efficiency for malignant glioma, providing promising tools for gene therapy.

2. Gene therapy for cancer

    a) Highly augmented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of glioma.  (Shinoura et al. Cancer Res. 59(14):3411-3416, 1999)  An E1B 55-kD gene-defective adenovirus has been reported to be a highly useful replication-competent adenovirus (Adv) which shows cytopathic effect for cancers with abnormality of p53 gene without damaging normal tissues.  In this study, we combined this adenovirus (Adv-E1AdB) with a fiber mutantion, F/K20 (Adv-E1AdB-F/K20), which has a stretch of 20 lysine residues added at the C-terminus of the fiber and shows high transduction efficiency to gliomas (Yoshida et al).  In U-373MG glioma cells, Adv-F/K20 for lacZ showed 9 times higher transduction efficiency than that of Adv with wild-type fiber (Adv-F/wt) for lacZ.  At the multiplicity of infection (MOI) of 30, Adv-E1AdB-F/K20 had 11 times higher replication efficiency compared with that of Adv-E1AdB with wild-type fiber (Adv-E1AdB-F/wt).  The in vitro cytopathic effect of Adv-E1AdB-F/K20 to U-373MG cells increased 32 times stronger in ED₅₀ value than that of Adv-E1AdB-F/wt.  The injection of Adv-E1AdB-F/K20 suppressed the in vivo growth of tumors; The anti-tumoral effect was remarkably stronger than that of Adv-E1AdB-F/wt.  Gene therapy utilizing Adv-E1AdB-F/K20, which drastically augmented the anti-tumoral effect of Adv-E1AdB, would be promising therapeutic approach for gliomas.

    b) Adenovirus-mediated transfer of p33ING1 with p53 drastically augments apoptosis in gliomas. (Cancer Res 59(21):5521-5528, 1999)  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.

3. Regenerative medicine.

    a) Stem cell biology: Long-term lymphohematopoietic reconstitution by a single CD34low/- hematopoietic stem cell  (Osawa et al.  Science, 273: 242-245, 1996.)  Hematopoietic stem cells (HSCs) supply all blood cell throughout life, by making use of their self-renewal and multilineage differentiation capabilities.  A monoclonal antibody (mAb) was raised to the mouse homologue of CD34 (mCD34), and was used to purify mouse HSCs to near homogeneity.  Unlike human, primitive adult mouse bone marrow (BM) HSCs were found in the mCD34 low to negative fraction.  Injection of a single mCD34low/-, c-Kit+, Sca-1+, lineage markers negative (Lin-) cell resulted in long-term reconstitution of the lymphohematopoietic system in 21% of recipients.  Thus, the purified HSC population will enable analysis of the self-renewal and multilineage differentiation of individual HSCs.

List of Main Publications from 1998 to 2000

１．        Shibagaki, N., Hanada, K., Yamaguchi, S., Yamashita, H., Shimada, S. and Hamada, H.  Functional analysis of CD82 in the early phase of T cell activation:  roles in cell adhesion and signal transduction.  Eur. J. Immunol., 28(4): 1125-1133, 1998.

２．        Shinoura, N., Yoshida, Y., Sadata, A., Hanada, K., Yamamoto, S., Kirino, T., Asai, A., and Hamada, H.  Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells:  implication for gene therapy.  Human Gene Therapy  9(14) : 1983-1993, 1998.

３．        Yoshida, Y., Sadata, A., Zhang, W., Shinoura, N. and Hamada, H.  Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma. Human Gene Therapy  9(17) : 2503-2515, 1998.

４．        Shinoura, N., Ohashi, M., Yoshida, Y., Asai, A., Kirino, T., Saito, I., and Hamada, H.  Construction, propagation, and titer estimation of recombinant adenoviruses for pro-apoptotic genes.  Human Gene Therapy  9(18) : 2683-2689, 1998.

５．        Shinoura, N., Yoshida, Y., Asai, A., Kirino, T., and Hamada, H.  Relative level of expression of Bax and Bcl-XL determines the cellular fate of apoptosis/necrosis induced by the overexpression of Bax. Oncogene, 18(41): 5703-5713, 1999.

６．        Shinoura, N., Yoshida, Y., Tsunoda, R., Ohashi, M., Zhang, W., Asai, A., Kirino, T., and Hamada, H.  Highly augmented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of glioma.  Cancer Res. 59(14):3411-3416, 1999.

７．        Shinoura, N., Yoshida, Y., Nishimura, M., Muramatsu, Y., Asai, A., Kirino, T., and Hamada, H.   Expression level of Bcl-2 determines anti- or pro-apoptotic function. Cancer Res. 59(16): 4119-4128, 1999.

８．        Shibagaki, N., Hanada, Yamashita, H., Shimada, S. and Hamada, H.  Overexpression of CD82 on human T cells enhances LFA1/ICAM1-mediated cell-cell adhesion: functional association between CD82 and LFA1 in T cell activation.  Eur. J. Immunol. 29(12):4081-4091., 1999.

９．        Shinoura, N., Muramatsu, Y., Nishimura, M., Yoshida, Y., Saito, A., Yokoyama, T., Furukawa, T., Horii, H., Hashimoto, M., Asai, A., Kirino, T., and Hamada, H.    Adenovirus-mediated transfer of p33ING with p53 drastically augments apoptosis in gliomas.  Cancer Res. 59(21):5521-5528, 1999.

１０．  Motoi, F., Sunamura, M., Ding, L., Duda, DG., Yoshida, Y., Zhang, W-P., Matsuno, S., and Hamada, H. Effective gene therapy for pancreatic cancer by cytokines mediated by restricted replication-competent adenovirus.  Human Gene Ther. 11(2): 223-235, 2000.

１１．  Shinoura, N., Yoshida, Y., Asai, A., Kirino, T., and Hamada, H.  Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas: Implication for gene therapy.  Hum. Gene Ther 11(8):1123-1137, 2000.