1. Volume 6, Issue 3, Pages 386-393 (September 2002)
Adenoviral Vectors with E1A Regulated by Tumor-Specific Promoters Are Selectively Cytolytic for Breast Cancer and Melanoma 
Lixin Zhang, Hakan Akbulut, Yucheng Tang, Xueyuan Peng, Giuseppe Pizzorno, Eva Sapi, Sebastian Manegold, Albert Deisseroth 
Molecular Therapy 
Volume 6, Issue 3, Pages (September 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Organization of conditionally replication-competent adenoviral vectors. The 2.5-kb L-plastin promoter or the human tyrosinase promoter and tissue-specific enhancer were inserted 5’ to the E1A and E1B genes. The regulatory elements left in the promoter in the adenoviral vectors are identical to those reported by Rodriguez et al. [4]. The viral packaging signal, which overlaps the E1A enhancer, has also been left in the area 5’ to the E1A transcription unit. Finally, it should be emphasized that both the E1A and E1B genes have been left in the vector.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Analysis of E1A expression and function. (A) E1A protein production in cells infected with Ad-Lp-E1A (lanes 1–4) and Ad-CMV-E1A (lanes 5–8). Ad5E1A protein expression in Yusac2 human melanoma cell line (lanes 1 and 5), TF2 human melanoma cell line (lanes 2 and 6), MDA-MB-468 human breast cancer cell line (lanes 3 and 7), and normal human mammary epithelial cells (lanes 4 and 8) was detected by western blot analysis. The molecular weight of the E1A protein marker is 46 kDa. We added 10 μg of protein to each lane. (B) The replication of the viral vectors (Ad-Lp-E1A, Ad-Tyr-E1A, and Ad-CMV-E1A) in different cell lines (293, Ovcar-5, MDA-MB-486, TF2, and Yusac2). Cell monolayers (2 × 105 cells/well) in six-well plates were inoculated with 2 × 105 pfu of the adenoviral vectors. The determination of the yield of vector (plaque titer) was carried out 72 hours after infection by the plaque assay on 293 cells in six-well plates.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Study of the sensitivity of breast cancer cells to the cytotoxic effect of the Ad-Lp-E1A vector. The MDA-MB-468 breast cancer cell line was exposed to the Ad-Lp-E1A vector at an MOI of 100 (left) and at an MOI of 10 (middle). The cells in the right panel were not exposed to vector. The pictures were taken 9 days after infection.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Study of the sensitivity of normal human mammary epithelial cells (HMEC) to the cytotoxic effect of the Ad-Lp-E1A vector. Normal HMEC cells were exposed to the Ad-Lp-E1A vector (top left), to the Ad-CMV-E1A vector (top right), and to wild-type adenovirus (bottom) at an MOI of 10 for 9 days.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Study of the sensitivity of explant cultures of human ovarian cancer cells to the cytotoxic effect of the Ad-Lp-E1A vector 9 days after infection. Explant cultures of human ovarian cancer cells were exposed to the Ad-Lp-E1A vector at MOIs of 1 and 10 (middle and right, respectively) or were not exposed to the vector (left).
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Study of the sensitivity of explant cultures of ovarian cancer cells to adenoviral vectors. Monolayer explant cultures of ovarian cancer cells were exposed to varying MOIs of the Ad-Lp-E1A vector, of the Ad-CMV-E1A vector, of the Ad-Tyr-E1A vectors, and of the replication-incompetent Ad-CMV-LacZ negative control vector. The percentage of cells killed was measured by the MTT assay [15] 7 days after infection. The percentage of cells killed is calculated as the ratio of the absorbance at 490 nm in the test culture to that of the culture without vector added. All data are the result of triplicate assays, and the standard deviation is less than 15%.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Cytotoxicity of the adenoviral vectors to the melanoma TF-2 cells 9 days after infection. Monolayers of the human melanoma cell line TF-2 were exposed to wild-type virus (top left), the Ad-CMV-E1A vector (bottom left), the Ad-Lp-E1A vector (top right), and the Ad-Tyr-E1A vector (bottom right).
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
The treatment breast cancer xenografts with adenoviral vectors. (A) Photograph of mice injected intratumorally with either the Ad-Lp-E1A vector (left) or with the Ad-CMV-LacZ vector (right) following injection of MCF-7 breast cancer cells. (B) The treatment of MCF-7 breast cancer xenografts with recombinant adenovirus. Tumor xenografts were treated with the recombinant vectors (108 pfu) Ad-Lp-E1A, wild-type adenovirus, and Ad-CMV-LacZ, and with PBS buffer by intratumoral injection on day 0 (1 week after tumor subcutaneous incubation) and measured. The data represent the mean of five replicate animals. The bars indicate the standard deviation about the mean of these values. The standard deviations about the mean (vertical error bars) of the sizes of the tumor nodules in animals injected with the negative controls (PBS or Ad-CMV-LacZ) and the test vectors (Ad-Lp-E1A or wild-type adenovirus) are statistically significantly different at the level, by the unpaired t-test. (C) The survival analysis of SCID mice with MDA-MB-486 xenografts. To establish subcutaneous tumor nodules, mice were inoculated subcutaneously in the rear flank with 5 × 106 MDA-MB-468 cells in 100 μl of PBS. The tumor growth was assessed by measuring bidimensional perpendicular diameters. Each animal was injected intratumorally with 109 pfu of the following viral vectors 1 week after inoculation of the MDA-MB-468 breast cancer cells: Ad-Lp-E1A, Ad-Tyr-E1A, wild-type adenovirus, and Ad-CMV-LacZ. The animals were killed by isoflurane when the tumors reached a volume of > 1000 mm3.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

10. FIG. 9
The treatment of TF-2 melanoma tumor xenografts with recombinant adenoviral vectors. The tumor xenografts were injected intratumorally with 108 pfu of Ad-Tyr-E1A, wild-type adenovirus, or Ad-CMV-LacZ, or by PBS buffer, by intratumoral injection on day 0 (1 week after subcutaneous tumor cell inoculation) and measured weekly. The data represent the mean of five replicate animals. The bars represent the standard deviation about the mean. The standard deviations about the mean of the tumor sizes in animals injected with either PBS or Ad-CMV-LacZ were not found to be statistically significantly different with the standard deviation about the mean of the sizes of tumor nodules in animals injected with the Ad-Tyr-E1A vector (P = or ) by the unpaired t-test.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions