Volume 4, Issue 1, Pages 13-21 (July 2001) Variation in Adenovirus Receptor Expression and Adenovirus Vector-Mediated Transgene Expression at Defined Stages of the Cell Cycle  Michael A. Seidman, Suzanne M. Hogan, Rebecca L. Wendland, Stefan Worgall, Ronald G. Crystal, Philip L. Leopold  Molecular Therapy  Volume 4, Issue 1, Pages 13-21 (July 2001) DOI: 10.1006/mthe.2001.0414 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Variability in Ad vector binding and transgene expression in A549 cells. Cells were infected with Cy3–Ad (1011 particles/ml, 10 min, 37°C) in binding buffer, washed, fixed, and assessed with primary antibody to cyclin B1 (mitosis) or cyclin E (G1/S transition) followed by an Oregon-Green-conjugated secondary antibody. Nuclei were stained with DAPI. Nuclear position, cyclin expression, and Cy3–Ad binding were evaluated by fluorescence microscopy. (A) Example of enhanced accumulation of Cy3–Ad in a cell undergoing mitosis. The top row of images shows a single microscopic field with staining for nuclei, cyclin B1, and Cy3–Ad. The bottom row shows a single microscopic field with staining for nuclei, cyclin E, and Cy3–Ad. One mitotic cell in the top row (arrow) contains condensed chromatin, stains for cyclin B1, and has a high concentration of bound Cy3–Ad. In the bottom row, one cell with uncondensed chromatin (arrow) stains for cyclin E (G1/S transition) but does not contain a high concentration of Cy3–Ad, whereas the same field includes one cell that is negative for cyclin E staining and bound a high concentration of Cy3–Ad (arrowhead). (B) Quantitative analysis of Cy3–Ad fluorescence intensity. Mitotic cells (n = 18) and interphase cells (n = 85) were evaluated by measuring the Cy3 fluorescence intensity per cell. Mitotic cells bound significantly higher levels of Cy3 compared with interphase cells (P < 0.02). Bar = 10 µm. Molecular Therapy 2001 4, 13-21DOI: (10.1006/mthe.2001.0414) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Pharmacological block of the cell cycle. Adherent A549 lung epithelial cells were cultured for 24 h before treatment with paclitaxel (0.1 µM), nocodazole (1.0 µM), or aphidicolin (5 µg/ml). To evaluate cell viability and growth kinetics, cells were incubated for an additional 48-h recording cell number at 24-h intervals. To evaluate distribution of stages of the cell cycle reflected in the population of cells, cells were collected after 24-h exposure to each compound, stained with propidium iodide, and analyzed for DNA content by flow cytometry. (A) Growth curves for control (treated with the drug diluent, DMSO) and cells treated with paclitaxel, nocodazole, or aphidicolin. (B) Flow cytometry scans of control, + paclitaxel (M block), + nocodazole (M block), and + aphidicolin (G1/S block) showing distribution of diploid cells in the G1 phase of the cell cycle (2N), tetraploid cells in the G2 and M phases of the cell cycle (4N), and an intermediate population of cells in the process of DNA synthesis (S phase). Molecular Therapy 2001 4, 13-21DOI: (10.1006/mthe.2001.0414) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Fluorescence microscopy analysis of adenovirus binding following treatment with cell cycle inhibitors. A549 cells were plated and treated with cell cycle inhibitors as described in Fig. 2. After 24 h, the cells were washed to remove the inhibitor, incubated for 1 h in culture medium, and infected with Cy3-conjugated Ad vector (1011 particles/ml, 10 min, 37°C). After infection, cells were washed, fixed, and analyzed by fluorescence microscopy. Single optical sections show Cy3–Ad in the red channel and DAPI-stained nuclei in the blue channel. (A) Control. (B) Paclitaxel (0.1 µM). (C) Nocodazole (1.0 µM). (D) Aphidicolin (5 µg/ml). Note that with paclitaxel and nocodazole, there is accumulation of Cy3–Ad on cells. Bar, 10 µm. Molecular Therapy 2001 4, 13-21DOI: (10.1006/mthe.2001.0414) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Flow cytometric analysis of adenovirus binding to A549 cells following treatment with cell cycle inhibitors. A549 cells were plated and treated with cell cycle inhibitors as described in Fig. 1; infection with Ad was as described in Fig. 2, except carboxyfluorescein-conjugated Ad vector (green fluorophore) was substituted for Cy3-conjugated Ad vector (red fluorophore). Cells were evaluated by flow cytometry. (A) Flow cytometry data from representative experiments include uninfected A549 cells compared with cells infected with carboxyfluorescein–Ad infected A549 cells; carboxyfluorescein–Ad infected control A549 cells compared with carboxyfluorescein–Ad infected, paclitaxel-treated A549 cells; carboxyfluorescein–Ad infected control A549 cells compared with carboxyfluorescein–Ad infected, nocodazole-treated A549 cells; and carboxyfluorescein–Ad infected control A549 cells compared with carboxyfluorescein–Ad infected, aphidicolin-treated A549 cells. (B) Quantification of flow cytometry. Data are presented as mean fluorescence intensity as a percentage relative to unsynchronized control cells. Molecular Therapy 2001 4, 13-21DOI: (10.1006/mthe.2001.0414) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Adenovirus-mediated gene transfer to cells following treatment with cell cycle inhibitors. A549 cells were plated and treated with cell cycle inhibitors as described in Fig. 2. After either 1 h (white bars) or 24 h (black bars), the cells were washed to remove drugs and infected with replication-deficient Adβgal vector (1011 particles/ml, 10 min, 37°C). After an additional 24 h, the cells were analyzed for β-galactosidase activity per mg protein. The data are presented as a % of β-galactosidase activity in control cells at each time point. Molecular Therapy 2001 4, 13-21DOI: (10.1006/mthe.2001.0414) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Flow cytometric analysis of cell surface coxsackie-adenovirus receptor (CAR) and αv integrin expression following treatment with cell cycle inhibitors. A549 cells were treated with cell cycle inhibitors as described in Fig. 2. After 24 h, the cells were stained with primary antibody (mouse anti-CAR or mouse anti-αv integrin) or an irrelevant primary antibody (Control) and secondary antibody (fluorescein-conjugated goat anti-mouse), and analyzed by flow cytometry. Data are presented as the percent change in mean fluorescence relative to control cells. Molecular Therapy 2001 4, 13-21DOI: (10.1006/mthe.2001.0414) Copyright © 2001 American Society for Gene Therapy Terms and Conditions