Young-Hwa Cho

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over-expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoters cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Inhibitory effects of the aqueous extract of Magnolia officinalis on the responses of human urinary bladder cancer 5637 cells in vitro and mouse urinary bladder tumors induced by N‐Butyl‐N‐(4‐hydroxybutyl) nitrosamine in vivo

This study investigated the anticancer activity of Magnolia officinalis on urinary bladder cancer in vitro and in vivo, and elucidated the mechanism of its activity. An aqueous extract of M. officinalis inhibited cell viability and DNA synthesis in cultured human urinary bladder cancer 5637 cells. Inhibition of proliferation was the result of apoptotic induction, because FACS analyses of 5637 cells treated with M. officinalis showed a sub‐G1 phase accumulation. M. officinalis extract also increased cytoplasmic DNA–histone complex dose‐dependently. These inhibitory effects were associated with the upregulation of proapoptotic molecules Bax, cytochrome c and caspase 3. Treatment of 5637 cells with M. officinalis extract suppressed the expression of matrix metalloproteinase 2 (MMP‐2) and MMP‐9, as revealed by zymographic and immunoblot analyses. When M. officinalis extract was given to mice simultaneously with the carcinogen N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine, which induces urinary bladder tumors, the size of the induced tumors was smaller. Finally, histological data indicated that the histological grade of carcinoma and the depth of invasion were dramatically decreased by treatment with M. officinalis extract in mice with N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine‐induced urinary bladder tumors. In conclusion, the findings showed that M. officinalis extract exhibited potential chemopreventive activity against urinary bladder tumor in vitro and in vivo. Copyright

Improved Production Efficiencies of Various Adeno-Associated Virus (AAV) Serotypes and a Novel Universal AAV Titration Method

Adeno-associated virus (AAV) has been considered to be a very safe and efficient gene delivery system. However, the major obstacles to therapeutic usage of AAV have been to achieve highly efficient and reproducible production processes, and also to develop a reliable quantifying method of various serotypes with a simple protocol. We compared the efficiency of the conventional production protocol of AAV2 and adenovirus (Ad) co-infection to that of a new method containing AAV2 infection followed by pHelper transfection. We tested HEK293 and 293T, and further examined the time-dependent changes of AAV2 production. The new method of AAV2 and pHelper DNA gave about ten times higher production efficiency than that of the conventional protocol. The highest production efficiency in 293T was achieved as 1.61 × 10? virus genomes (v.g.)/cell by the new method of 10 MOI of AAV2 infection and 5 days post-infection. This protocol of the highest efficiency was then applied to produce various AAV serotypes and showed the efficiencies higher than 10? v.g./cell. Next, we designed the universal PCR primers of highly conserved regions for various AAV serotypes to develop a simple and reliable titration method. The universal primers could amplify all the tested AAV serotypes with similar sensitivities by ten molecular copies. Therefore, this pair of universal primers can be further utilized to detect AAV contaminants in therapeutic adenoviral vectors.

A Cancer-specific Promoter for Gene Therapy of Lung Cancer, Protein Regulator of Cytokinesis 1 (PRC1)

2 Division of Radiation Cancer, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul 139-706, Korea - We have recently re- ported the PRC1 promoter as a promoter candidate to control expression of transcriptionally targeted genes for breast cancer gene therapy. We tested whether the PRC 1 promoter could be also applied for the lung cancer gene therapy. In the transient transfection assay with naked plasmids containing the luciferase fused to the PRC1 promoter, the promoter showed little activity in the normal lung cell line, MRC5. However, in the lung cancer A549 cells, PRC1 showed approximately 30-fold activa- tion which was similar to the survivin promoter, the gene whose promoter has been already re- ported as a candidate for the gene therapy of lung cancer. In viral systems, the PRC1 promoter showed approximately 75% and 66% of transcriptional activity compared to the CMV promoter in the adeno-associated virus (AAV) and the adenovirus (AV) systems, respectively. However, the PRC1 promoter in either AAV or AV showed approximately 20% activity compared to the CMV promoter in the normal lung cells. In addition, human lung tumor xenograft mice showed that the PRC1 promoter activity was as strong as the CMV activity in vivo. Taken together, these results sug- gested that PRC1 might be a potential promoter candidate for transcriptionally targeted lung cancer gene therapy.