This step is possible only through the metaphasic breakdown of the nuclear membrane [14, 16, 30].

Therefore, the integration of retroviral DNA during cell division has only been evidenced Selleckchem NVP-BGJ398 when the doubling time of target cells was higher than the half-life of the virus [15]. As the half-life of MuLV-derived vectors is between 5.5 and 7.5 hr [31] and as the ACY-1215 cost DHDK12 and HT29 cell lines have a doubling time of 28 hr [32] and 24 hr [33], respectively, our model meet this criterion. Our experimental design thus was adapted to study the efficiency of retroviral gene transfer after pharmacological control of the cell cycle. Cell synchronization has been used to increase the number of cells accessible to drug targeting DNA and to improve the action of several anti-proliferative chemotherapies [20, 23, 24]. In this regard, experimental works have studied the synchronization

in S phase of cancer cell lines Smoothened Agonist order by MTX, aphidicolin or ara-C. Aphidicolin and ara-C are reversible inhibitors of DNA polymerases [18, 22]. MTX induces a reversible inhibition of dihydrofolate reductase, which is required for the de novo synthesis of nucleotides for DNA replication [34]. Our study showed a limited efficiency of ara-C or aphidicolin in DHDK12 cells. Moreover, a significant toxicity of aphidicolin, not compatible with an in vivo application, has been observed on several cancer cell lines [19, 35]. We observed that non-toxic concentrations SPTLC1 of MTX induced a reversible synchronization of DHDK12 and HT29 cells in early S phase (Figure 1). A 24 hr-treatment with MTX allowed increasing the rate of cells in S phase. The reversibility of MTX was confirmed as the cells returned to the normal cell cycle according

to there doubling time. These results were in accordance to those obtained in others cell lines [36]. The reverse transcription of retroviral DNA can occur in several phases of the cell cycle [16]. However, the cells should be stimulated to divide before infection for efficient gene transfer [37]. According to the intracellular half-life of retroviral intermediates, the position of target cells relative to mitosis and the duration of S phase at the time of exposure both are critical to determine the efficiency of infection [38]. This assumption was supported by the difference in retroviral gene transfer improvement between DHDK12 and HT29 cell lines after cell synchronization by MTX. These two colon cancer cell lines exhibit a different pattern of cell cycle distribution after synchronization (Figure 1). We have observed that in HT29 cells the level of transgene expression, which was lower than that observed in DHDK12 cells, was strictly related to the peak of cells in S phase (Figure 2B). In DHDK12 cell line, the peak of cells in S phase was located 10 hr after the recovery and the infection efficiency was improved by 2-fold 20 hr after MTX removal (Figure 2A).