Enhancement of apoptosis in synchronized cell To determine whether the improvement of HSV-tk gene transfer efficiency by cell synchronization resulted into an increased GCV-mediated cell death, we selleck chemicals measured the level of cell apoptosis after GCV treatment using annexin V-FITC. The presence of apoptosis observed with annexin V labeling was confirmed by the DNA fragmentation method (Figure 5). Annexin V labeling

was increased in MTX-treated DHDK12 and HT29 cells transduced with HSV-tk gene and then treated for 72 hr by GCV. Figure 5 Internucleosomal PX-478 DNA fragmentation induced by GCV. Lane 1 and lane 4 show DHDK12 cells and HT29 cells transduced with TG 9344 and treated for 96 hr with 20 μM GCV, respectively. Lane 3 and 5 show DHDK12 cells and HT29 cells transduced with TG 9344 after a 24 hr pretreatment with MTX and treated for 96 hr with 20 μM GCV, respectively. Lane 6 and 7 show DHDK12 cells and HT29 cells treated for 24 h with MTX, respectively. Lane 2 shows pBR 322 base pair size markers. Qualitative detection of DNA was achieved by ethidium bromide staining. In non-transduced cells, 5% of

MTX treated cells Berzosertib cost were labeled for annexin V-FITC after treatment by GCV (Figure 6A). This corresponds to the intrinsic toxicity of MTX. Figure 6 Induction of apoptosis. Untransduced DHDK12 cells (A) were treated with MTX, GCV or the combination of MTX plus GCV for 24 h. Transduced DHDK12 cells (B) and transduced HT29 cells (C) were treated for 24 hr with (filled Cyclin-dependent kinase 3 square) or without (open square) MTX. Cells were transduced with TG 9344 at the indicated times after MTX washout and 48 hr after transduction were treated with 20 μM GCV for 72

hr. Quantitative detection of apoptosis was determined by biparametric flow cytometry analysis of fluorescein labeled-annexin V cells and PI. Apoptotic cells were annexin V positive, PI negative. Data are expressed as the mean ± SE from at least three separate experiments. * P <.05 vs untreated cells The percentage of MTX-treated DHDK12 cells undergoing apoptosis (Annexin V+, PI-) was two fold higher after MTX withdrawal (46% vs. 23% in the untreated cell population). The difference was maximal in cells transduced 20 hr after MTX withdrawal (Figure 6B). In HT29 cells, the maximum percentage of MTX-treated cells undergoing apoptosis was 28% while it was 20% in untreated cells. The highest level of cell apoptosis was maximal 6 hr after MTX withdrawal (Figure 6C). Discussion The objective of this work was to improve the efficiency of retroviral transfer of the suicide gene HSV-tk in colon cancer cells. This aim was achieved through the pharmacological control of the target cells cell cycle. Our results are consistent with previous reports showing that retroviral-mediated gene transfer depends on the cell cycle of target cells.