Correlation between the radiosensitising effect of vinflunine (VFL) and its cell cycle effects, in different treatment schedules Free

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VFL proved to have radiosensitising potential and caused a clear G2/M blockade (Simoens et al., CCP, in press). However, further investigation was necessary to elucidate the exact role of this cell cycle effect. In this study, the cell cycle effects of VFL were correlated to its radiosensitising effect, using different treatment schedules. ECV304, a bladder cancer, and CAL-27, a head and neck cancer cell line were treated with radiosensitising VFL concentrations (50 nM and 20 nM, resp.). To investigate the influence of the incubation time, cells were treated during 8, 24 or 48h prior to radiation (RT) (Co-60γ rays, 0-8 Gy, room temp.). The influence of a time interval was tested by 24h incubation with VFL, 0, 8 or 24h before RT (24+0, 24+8, 24+24). Cell survival was determined by the SRB assay 7 or 8 days after RT and radiosensitisation (RS) was represented by the Dose Enhancement Factor (DEF). To determine the role of the cell cycle perturbations caused by VFL at the moment of RT, cells were analysed by flow cytometry (DNA staining). Cells were treated with 150 nM (ECV304) and 100 nM VFL (CAL-27) using the above mentioned treatment schedules. In both ECV304 and CAL-27, 8h of incubation did not result in a radiosensitising effect, although a significant increase in G2/M phase cells was observed. This increase was probably not strong enough to compensate for the simultaneous significant increase in S phase cells (most radioresistant) at that time. Maximum RS was reached after 24h incubation (max. G2/M block) and decreased with longer incubation times (48h), which coincided with a decrease of G2/M phase cells and an increased polyploid population. Using different time intervals, maximum RS (DEF=2,01) was observed after 24h incubation immediately followed by RT (24+0) in ECV304. This also resulted in the largest arrest at G2/M (57,3% v. 17,8% in control cells). RS gradually decreased with an increasing time interval between VFL treatment and RT, which coincided with a decrease in G2/M, an increase in G1 (24+8) and an increase in the polyploid population (24+24). In CAL-27, RS was most pronounced after an 8h interval between VFL treatment and radiation (24+8) (DEF=2,36 v. 1,79 (24+0)). The only linked cell cycle effect was the lowest amount of S phase cells at that time, although the amount of G2/M cells was already significantly decreased compared to 24+0. 24+24 resulted in a strong decrease of RS, together with the highest amount of cells in S and the polyploid population. In conclusion, the radiosensitising effect of VFL after different treatment schedules is cell line-dependent. The observed G2/M block cannot explain the altered radiosensitivity all by itself. Other cell cycle effects, like the presence of S phase and polyploid cells, also play an important role in the radiosensitivity after VFL treatment. In addition, other non-cell cycle-related causes may exist.