小澤 敬也

Adeno-associated virus type 2 integrates preferentially into the AAVS1 locus on chromosome 19 of the human genome. It was reported previously that transfection with two plasmids, one for Rep and the other carrying a transgene flanked by inverted terminal repeats (ITRs), enables preferential integration of the latter into AAVS1. Aiming at increasing the frequency of AAVS1-specific integration, the Rep- to transgene-plasmid ratio necessary to achieve a higher frequency of site-specific integration was examined. 293 cells were co-transfected with the Rep78 plasmid and an ITR-flanked Neo gene at different ratios. G418-resistant clones were selected randomly. Extensive Southern blot analysis showed an optimum range of Rep78 expression. In that range, approximately 20% of clones harboured the Neo gene at AAVS1. Excess Rep expression, however, resulted in 'abortive' integration of the Neo gene, a rearrangement of AAVS1 without transgene integration. Rep78 appeared to cause abortive integration more extensively than Rep68. Deleterious effects of the Rep protein on the AAVS1 locus should be considered to develop an improved AAVS1-targeted system.

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibiior of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting intheprotection of erythroid cells from apoptosis. J. Cell. Physiol. 195: 290-297, 2003. (C) 2003 Wiley-Liss, Inc.

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibiior of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting intheprotection of erythroid cells from apoptosis. J. Cell. Physiol. 195: 290-297, 2003. (C) 2003 Wiley-Liss, Inc.

Ideno, J., Mizukami, H., Honda, K., Okada, T., Hanazono, Y., Kume, A., Saito, T., Ishibashi, S., and Ozawa, K.: Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector-expressing arginine-vasopressin in brattleboro rats.

Ishii, H., Vecchione, A., Furukawa, Y., Sutheesophon, K., Han, S.Y., Druck, T., Kuroki, T., Trapasso, F., Nishimura, M., Saito, Y., Ozawa, K, Croce, C.M., Huebner, K., and Furukawa, Y.: Expression of FRA16D/WWOX and FRA3B/FHIT genes in hematopoietic malignancies.

Nagata, M., Takahashi, M., Muramatsu, S., Ueda, Y., Hanazono, Y., Takeuchi, K., Okada, K., Suzuki, Y., Kondo, Y., Suemori, M., Ikeda, U., Nakano, I., Kobayashi, E., Hasegawa, M., Ozawa, K., Nakatsuji, N., and Shimada, K.: Efficient gene transfer of a simian immunodeficiency viral vector into cardiomyocytes derived from primate embryonic stem cells.

Asano, T., Ageyama, N., Takeuchi, K., Momoeda, M., Kitano, Y., Sasaki, K., Ueda, Y., Suzuki, Y., Kondo, Y., Torii, R., Hasegawa, M., Ookawara, S., Harii, K., Terao, K., Ozawa, K., and Hanazono, Y.: Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells.

Oshima, Y., Ueda, M., Yamashita, Y., Choi, Y.L., Ota, J., Ueno, S., Ohki, R., Koinuma, K., Wada, T., Ozawa, K., Fujimura, A., and Mano, H.: DNA microarray analysis of hematopoietic stem cell-like fractions from individuals with the M2 subtype of acute myeloid leukemia.

Shibata, H., Hanazono, Y., Ageyama, N., Nagashima, T., Ueda, Y., Hasegawa, M., Ozawa, K., Yoshikawa, Y., and Terao, K.: Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): Assessment of cross-reacting monoclonal antibodies.

Kohno, T., Mizukami, H., Suzuki, M., Saga, Y., Takei, Y., Shimpo, M., Matsushita, T., Okada,T., Hanazono, Y., Kume, A., Sato, I., and Ozawa, K.: Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGF-producing ovarian cancer.

Komatsu, N., Watanabe, T., Uchida, M., Mori, M., Kirito, K., Kikuchi, S., Liu, Q., Tauchi, T., Miyazawa, K., Endo, H., Nagai, T., and Ozawa, K.: A member of Forkhead transcription factor FKHRL1 is a downstream effector of STI571-induced cell cycle arrest in BCR-ABL expressing cells.

Lu, Y.-Y, Wang, L.J., Muramatsu, S., Ikeguchi, K., Fujimoto, K., Okada, T., Mizukami, H., Matsushita, T., Hanazono, Y., Kume, A., Nagatsu, T., Ozawa, K., and Nakano, I.: Intramuscular injection of AAV-GDNF results in sustained expression of transgenic GDNF, and its delivery to spinal motoneurons by retrograde transport.

Kanazawa, T., Mizukami, H., Okada, T., Hanazono, Y., Kume, A., Nishino, A., Takeuchi, K., Kitamura, K., Ichimura, K., and Ozawa, K.: Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice.

Ideno, J., Mizukami, H., Honda, K., Okada, T., Hanazono, Y., Kume, A., Saito, T., Ishibashi, S., and Ozawa, K.: Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector-expressing arginine-vasopressin in brattleboro rats.

Ishii, H., Vecchione, A., Furukawa, Y., Sutheesophon, K., Han, S.Y., Druck, T., Kuroki, T., Trapasso, F., Nishimura, M., Saito, Y., Ozawa, K, Croce, C.M., Huebner, K., and Furukawa, Y.: Expression of FRA16D/WWOX and FRA3B/FHIT genes in hematopoietic malignancies.<br />

Nagata, M., Takahashi, M., Muramatsu, S., Ueda, Y., Hanazono, Y., Takeuchi, K., Okada, K., Suzuki, Y., Kondo, Y., Suemori, M., Ikeda, U., Nakano, I., Kobayashi, E., Hasegawa, M., Ozawa, K., Nakatsuji, N., and Shimada, K.: Efficient gene transfer of a simian immunodeficiency viral vector into cardiomyocytes derived from primate embryonic stem cells.<br />

Asano, T., Ageyama, N., Takeuchi, K., Momoeda, M., Kitano, Y., Sasaki, K., Ueda, Y., Suzuki, Y., Kondo, Y., Torii, R., Hasegawa, M., Ookawara, S., Harii, K., Terao, K., Ozawa, K., and Hanazono, Y.: Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells.<br />

Oshima, Y., Ueda, M., Yamashita, Y., Choi, Y.L., Ota, J., Ueno, S., Ohki, R., Koinuma, K., Wada, T., Ozawa, K., Fujimura, A., and Mano, H.: DNA microarray analysis of hematopoietic stem cell-like fractions from individuals with the M2 subtype of acute myeloid leukemia.<br />

Shibata, H., Hanazono, Y., Ageyama, N., Nagashima, T., Ueda, Y., Hasegawa, M., Ozawa, K., Yoshikawa, Y., and Terao, K.: Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): Assessment of cross-reacting monoclonal antibodies.<br />

Kohno, T., Mizukami, H., Suzuki, M., Saga, Y., Takei, Y., Shimpo, M., Matsushita, T., Okada,T., Hanazono, Y., Kume, A., Sato, I., and Ozawa, K.: Interleukin-10-mediated inhibition of angiogenesis and tumor growth in mice bearing VEGF-producing ovarian cancer.<br />