1. Volume 6, Issue 2, Pages 190-198 (August 2002)
Adenovirus-Mediated Transfer of BAX Driven by the Vascular Endothelial Growth Factor Promoter Induces Apoptosis in Lung Cancer Cells 
Sergey A Kaliberov, Donald J Buchsbaum, G.Yancey Gillespie, David T Curiel, Waleed O Arafat, Mark Carpenter, Murray A Stackhouse 
Molecular Therapy 
Volume 6, Issue 2, Pages (August 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
EGFP expression in lung cell lines analyzed using FACS. Cells were harvested 48 hours after AdVEGFEGFP or AdCMVEGFP (5000 v.p./cell) infection. Samples (10,000 cells) were analyzed by FACScan. EGFP fluorescence was the mean fluorescence signal in EGFP+ cells in ru after subtraction of background fluorescence. Data expressed as percentage of EGFP+ cells are the means after subtraction of uninfected control cells. Presented are mean values ± SD of two to five independent experiments, each carried out in triplicate.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
VEGF protein and mRNA expression in lung cell lines. (A) The level of expression of VEGF protein was examined using ELISA. VEGF concentrations in conditioned media of the indicated cell lines were normalized to number of viable cells. Presented are mean values ± SD of experiments that were carried out in duplicate cultures in two separate experiments. (B) Analysis of VEGF mRNA transcripts by RT-PCR using primers specific for all known isoforms. The size of the indicated bands of 441 and 573 bp was determined by a 100-bp DNA ladder and correspond to VEGF121 and VEGF165, respectively. One representative of three different experiments is shown.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
AdVEGFBAX preferentially induces death in lung cancer cells. (A) Normal human bronchial epithelium cells (BEAS-2B) and lung carcinoma (H1466 and A427) cells were infected with the recombinant adenoviruses as indicated. Cell viability was determined at 96 hours after infection by using the MTS assay. Data shown are mean values ± SD of three experiments. (B) Induction of BAX protein expression causes lung cancer cell death. Cell viability after infection (5000 v.p./cell) of H1466 cells with AdVEGFBAX (closed columns) or AdVEGFEGFP (opened columns) was determined using Trypan Blue exclusion. The data show mean values ± SD of three experiments. (C) The level of expression of HA-BAX and β-actin was examined using a western blot technique. After AdVEGFBAX infection (5000 v.p./cell), H1466 cells were collected at the times indicated. Equal amounts (50 μg) of protein were loaded for each sample in all lanes and electrophoretically separated on 12% SDS–PAGE followed by transfer to a PVDF membrane. One representative of three different experiments is shown.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Apoptosis profile of H1466 cells after AdVEGFBAX infection. (A) For annexin-V binding assay and propidium iodide (PI) uptake evaluation, after AdVEGFBAX or AdVEGFEGFP infection (5000 v.p./cell) cells were collected over time as indicated and double-stained with FITC-conjugated annexin-V and PI. One representative of three different experiments is shown. (B) All cells taking vital dye PI were considered necrotic, annexin V+cells were considered apoptotic, and only cells in the lower right quadrant were used for calculation of percentages from the total number of cells. Samples (10,000 cells) were analyzed by FACScan. Data points are the mean values ± SD of two or three experiments, each conducted in triplicate.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Overexpression of BAX induces apoptosis in H1466 lung cancer cells through activation of multiple caspases. (A) The cleavage of caspases-3, -8 and -9 was monitored by western blot analysis. H1466 cells were collected 16 hours after AdVEGFBAX infection (5000 v.p./cell). Equal amounts (100 μg) of protein were loaded for each sample in all lanes and electrophoretically separated on 10% SDS–PAGE, followed by transfer to a PVDF membrane. One representative of three different experiments is shown. (B) Caspase activity was measured using Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC as fluorometric substrates for caspases-3, -8, and -9 respectively. After AdVEGFBAX or AdVEGFEGFP infection (5000 v.p./cell), cells were harvested over time as indicated. Samples were read in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. Fold increase in caspase activity was determined by comparing the result with the level of the uninfected control cells. Data shown are the mean ± SD.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Effect of hypoxic conditions on VEGF protein expression. (A) VEGF-specific ELISA was used to determine VEGF concentrations in conditioned media of the human lung cell lines incubated under normoxic (O2, 21%) or exposed to hypoxic (O2, 1%) conditions for 16 hours and normalized to number of viable cells. (B) Fold increase protein concentration was determined by comparing VEGF concentration in conditioned media after incubation under hypoxia with the level of the VEGF protein secretion under normoxic conditions. Data presented are mean values ± SD of two separate experiments. The VEGF promoter response to hypoxic conditions is determined. (C) For determination of response of VEGF promoter element to hypoxic conditions, following infection with AdVEGFEGFP (6000 v.p./cell), BEAS-2B human lung cells were cultured under normoxic (O2, 21%) or exposed to hypoxic (O2, 1%) conditions for 18 hours and after reoxygenation for 4 hours examined for EGFP expression by fluorescent microscopy. Original magnification was ×100. (D) At the same time points after AdVEGFEGFP infection, BEAS-2B and A549 cells were harvested and samples (10,000 cells) were analyzed by FACScan. EGFP fluorescence was the mean fluorescence signal in EGFP+ cells in ru after subtraction of background fluorescence. Data presented are mean values ± SD of three separate experiments.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
AdVEGFBAX enhances lung cancer cell death through increasing apoptosis under hypoxic conditions. (A) BEAS-2B and A549 lung cells after AdVEGFBAX (5000 v.p./cell) infection were exposed to hypoxic (O2, 1%) or cultured under normoxic (O2, 21%) conditions for 72 hours, and cell viability was determined using the MTS assay. Each bar represents the mean values ± SD of three experiments. (B) After infection with 5000 v.p./cell AdVEGFBAX, A549 and BEAS-2B cells were cultured under normoxic (O2, 21%) or exposed to hypoxic (O2, 1%) conditions for 48 hours and after reoxygenation for 4 hours were examined for annexin-V binding and PI uptake. Cells were collected and double-stained with FITC-conjugated annexin-V and PI. Cells taking up vital dye PI were considered necrotic; annexin-V+ cells were considered apoptotic and their percentages were calculated. Samples (10,000 cells) were analyzed by FACScan. Data expressed as percentage of annexin-V+ cells are the means after subtraction of uninfected control. Data presented are the mean values ± SD of three independent experiments.
Molecular Therapy 2002 6, DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions