1. Volume 3, Issue 5, Pages 642-652 (May 2001)
BCR-ABL-Expressing Cells Transduced with the HSV-tk Gene Die by Apoptosis upon Treatment with Ganciclovir 
Carmen Rivas, Angela R.-M Miller, Manuel Collado, Eric W.-F Lam, Jane F Apperley, Junia V Melo 
Molecular Therapy 
Volume 3, Issue 5, Pages (May 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
GCV sensitivity of transduced clones. Proliferation assays on transduced clones in the presence of several concentrations of GCV were assessed. Results are expressed as the percentage of the [3H]thymidine incorporation compared to that of each clone grown in the absence of GCV.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
GCV toxicity time course of transduced clones. The viability of tk-transduced TonB210.1 and 32D parental cells as well as Bcr-Abl-expressing clones was measured at different time points after treatment with 0.8 μg/ml GCV using trypan blue dye exclusion. The percentage viability was calculated based on the number of cells present at the initiation of GCV treatment. Results are representative of identical experiments with three clones from each cell line.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Growth curve of tít-transduced parental and Bcr-Abl-expressing cells in medium without GCV. 1 × 106 cells were incubated in triplicate in growth medium supplemented (parental cells) or not (cells expressing Bcr-Abl) with WEHI conditioned medium and the number of viable cells was counted at 1, 2, and 3 days. Results are representative of identical experiments with three clones from each cell line.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Treatment with GCV induces apoptosis of both tk-transduced 32D and TonB210.1 parental cells and Bcr-Abl-expressing cells. (a) DNA fragmentation in transduced clones after 2 days of incubation with 0.8 μg/ml GCV was assayed by fractionating equal amounts of genomic DNA on 1.6% agarose gels. DNA laddering was detected by ethidium bromide staining. Results are representative of identical experiments with three clones from each cell line. (b) Double annexin-V-FITC and PI staining of 32D/tk and 32Dp210/tk cells treated with GCV for the indicated times. Numbers on the lower and upper right quadrants represent the percentage of cells in the early stages of apoptosis and in the later stages of cell death, respectively.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Flow cytometric analysis of DNA content after propidium iodide staining of TonB210.1 cells treated with 0.8 μg/ml GCV. The numbers show the percentages of cells in the different phases of the cell cycle at the time of GCV addition (top), and after 10 h (second), 24 h (third), and 48 h (bottom) of drug exposure. The data are representative of three separate experiments.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Cell cycle of transduced clones after treatment with GCV. Transduced clones were grown in the presence of 0.8 μg/ml GCV for the times indicated. The percentages of cells in S, G2/M, and sub-G1 phase of the cell cycle were measured using flow cytometric analysis of DNA content after propidium iodide staining. The data are representative of three separate experiments.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
CD95 and Fas-L expression on transduced clones after treatment with GCV. Representative FACS profiles showing CD95 and Fas-L expression levels in transduced clones in the absence of GCV (bold line) and at 19 h after GCV treatment (gray line). Relative fluorescence values are shown on the x axis. Results are representative of identical experiments with three clones from each cell line.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

9. FIG. 8
Caspase-8 activation. Transduced clones were treated for 24 or 48 h with GCV and assayed for caspase-8 activation using the ApoAlert Flice/caspase-8 colorimetric assay kit. Results are expressed as the mean percentages of increase in DEVD-dependent protease activity in treated relative to untreated samples. The variations between duplicates were less than 10%.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

10. FIG. 9
Attenuation of apoptosis by the caspase-8 inhibitor Z-IETD-FMK. Cells were treated with 0.8 μg/ml GCV in the presence or absence of the caspase-8 inhibitor at 50 μM for 28 h (TonB210.1 and 32D cells) or 50 h (32Dp210 cells). Apoptosis was determined by flow cytometric analysis as described under Materials and Methods. The histograms represent the percentage of subdiploid cells in each cell culture at the indicated times after the treatment.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

11. FIG. 10
Effect of GCV treatment on Bcl-2 and Bcl-xL proteins. Cells were grown with 0.8 mg/ml GCV for the indicated times and analyzed by Western blot staining with the appropriate antibody. Actin was used as an internal control.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

12. FIG. 11
Downregulation of Bcr-Abl protein levels in tk-transduced clones after treatment with GCV. (A) Western blot analysis of the levels of Bcr-Abl in tk-transduced 32Dp210 and induced TonB210.1 clones at different times after GCV treatment. Actin was used as an internal control. (B) Representative FACS profiles showing Bcr-Abl expression levels in TonB210.1 cells (1) in the absence of doxycycline, (2) in the presence of doxycycline at time zero, (3) at 24 h, and (4) at 48 h after GCV treatment. Relative fluorescence values are shown on the x axis.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions