1. Volume 10, Issue 6, Pages 1043-1050 (December 2004)
Prolonged Survival of Mice with Multiple Liver Metastases of Human Colon Cancer by Intravenous Administration of Replicable E1B-55K-Deleted Adenovirus with E1A Expressed by CEA Promoter 
Tamotsu Sagawa, Minoru Takahashi, Tsutomu Sato, Yasushi Sato, Yue Lu, Tetsuya Sumiyoshi, Yasuyuki Yamada, Satoshi Iyama, Junki Fukaura, Katsunori Sasaki, Hirofumi Hamada, Koji Miyanishi, Tetsuji Takayama, Junji Kato, Yoshiro Niitsu 
Molecular Therapy 
Volume 10, Issue 6, Pages (December 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
A schematic diagram of the structure of the selectively replication-competent adenovirus-AdCEAp/Rep genome. The double expression cassette consists of E1A-13S under the control of a 493-bp fragment of the human CEA promoter and E1B-19K under the control of CMVp tandemly inserted into the E1A region.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Immunotransblot analysis of E1A-13S after infection of gastrointestinal carcinoma cell lines with AdCEAp/Rep. Cell homogenates were prepared as described under Materials and Methods. Immunoreactive 43-kDa bands, corresponding to human Ad5 E1A, were observed in M7609, MKN45, and BxPC3, but not in Hc or in PLC/PRF/5. The lanes show results using cell homogenates from the following sources: lane 1, Hc + NS; lane 2, Hc + AdCEAp/Rep; lane 3, PLC/PRF5 + NS; lane 4, PLC/PRF5 + AdCEAp/Rep; lane 5, M NS; lane 6, M AdAFPep/Rep; lane 7, MKN45 + NS; lane 8, MKN45 + AdAFPep/Rep; lane 9, BxPC3 + NS; lane 10, BxPC3 + AdAFPep/Rep. NS, normal saline.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Viral yield after infection with AdCEAp/Rep. MKN45, BxPC3, M7609, HT-29, P5, and Hc cells were infected with AdCEAp/Rep at 100 PFU/cell. Cells and supernatant were harvested 3 days after infection, and virus yield was measured by titration on 293 cells. Data shown are the mean results ± SD (bars) of a representative experiment performed in triplicate. PFU, plaque-forming unit; P5, PLC/PRF5.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Cytotoxic effect of AdCEAp/Rep. Five cancer cell types (MKN45, BxPC3, M7609, HT-29, and PLC/PRF5) and Hc were infected with AdCEAp/Rep at various m.o.i. Cytotoxicity was measured by a dye-uptake assay at day 7 postinfection. Data shown are the mean results ± SD (bars) of a representative experiment performed in triplicate. Hc, primary human hepatocytes; P5, PLC/PRF/5.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Cell survival after infection with AdCEAp/Rep or AdCEAp/nlacZ. M7609 and BxPC3 cells were infected with AdCEAp/Rep or AdCEAp/nlacZ at m.o.i. of 0.1. Cell survival was evaluated by a dye-uptake method at the indicated time points. Cell survival rates are presented as the percentages of uninfected cells at the same time points. Data shown are the mean results ± SD (bars) of a representative experiment performed in triplicate.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Antitumor effects of AdCEAp/Rep on M7609 tumor established in athymic nude mice. M7609 cells were inoculated into nude mice, and PBS (0.1 ml), AdCEAp/nlacZ (1 × 108 PFU/0.1 ml), or AdCEAp/Rep (1 × 108 PFU/0.1 ml) was administered into established tumors. Each experimental group contained six mice. The tumors of the treatment group were administered AdCEAp/Rep (1 × 108 PFU/0.1 ml) once on day 0. PBS or AdAFPep/nlacZ was injected on day 0 for the nontreatment group. (A) An example of the subcutaneous tumor before treatment and a previously established subcutaneous tumor at day 20 after treatment showing the effect of intratumoral injection of AdCEAp/Rep or AdCEAp/nlacZ. (B) Tumor growth suppression by intratumoral injection of AdCEAp/Rep. Tumor diameters were measured with calipers, and tumor volumes were calculated as described under Materials and Methods.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Effects of intravenous administration of AdCEAp/Rep on established liver metastases. Multiple liver metastases were established by administration of HT-29 human colon carcinoma cells via the spleen in a nude mouse model. At day 5 after tumor inoculation, a splenectomy was performed. Mice received intravenous injections containing AdCEAp/Rep, AdCEAp/nlacZ, or PBS on a daily basis from day 8, when multiple tiny spots of tumor metastases were microscopically confirmed in the other experimental group, to day 12 after inoculation of tumor. Mice were euthanized on day 43, and their livers were removed and weighed (n = 4). The effects of AdCEAp/Rep injection into the tail vein on (A) liver weight and (B) the number of metastatic foci on the surface of the liver after inoculation of tumor cells are shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

9. Fig. 8
Replication of AdCEAp/Rep in subcutaneous tumor and metastatic liver tumor. The DNA samples isolated from specimens were analyzed by real-time quantitative PCR for the adenovirus E4 copy number, and the results are shown as adenoviral copy number per 100 ng DNA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

10. Fig. 9
Survival of nude mice after systemic administration of AdCEAp/Rep. Kaplan–Meier survival curve for the mice of each treatment group (n = 10). Mice received AdCEAp/Rep, AdCEAp/nlacZ, or PBS iv on days 0–5. The mice treated with AdCEAp/Rep yielded a significantly higher survival rate (P = for the AdCEAp/nlacZ group; P = for the PBS group, n = 5).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions