Role of 2′-2′ difluorodeoxycytidine (gemcitabine)-induced cell cycle dysregulation in radio-enhancement of human head and neck squamous cell carcinomas

Abstract

Background and purpose To try to get a better insight on the interaction between dFdC and ionizing radiation at the cellular level, we examined in vitro the effect of dFdC on the cell cycle of two human head and neck squamous cell carcinoma cell lines (SQD9 and SCC61). Patients and methods Experimental conditions yielding radio-enhancement were used. Confluent cells were incubated with dFdC (5 μM) for different incubation times, washed, pulse-labeled with BrdUrd (10 μM), fixed and then processed for flow cytometry analysis. Alternatively, cells preincubated or not with dFdC were irradiated (5 Gy) in drug-free medium, incubated at 37 °C for various times and then processed for flow cytometry analysis. Results In both cell lines, dFdC incubated between 1 and 6 h induced a DNA synthesis inhibition with accumulation of cells in the G1–S boundary followed, when DNA reinitiated, by a synchronous progression of cells throughout the cycle. A slightly different kinetics was observed in the two cell lines. A weak correlation between dFdC radio-enhancement and distribution of cells in the cell cycle was observed. It was also observed that for longer dFdC incubation times, DNA synthesis could reinitiate while cells were still incubated with dFdC. This reinitiation could be correlated with a decrease in the intracellular dFdCTP pool to non-inhibitory levels. Finally in both cell lines, dFdC modified neither the importance nor the kinetics of the radiation-induced G1 delay. Conclusions This study provides evidence that gemcitabine used at radio-enhancing concentration induces alteration of cell kinetics and cell redistribution throughout the cell cycle. This effect is cell line-dependent. However, the weak correlation between dFdC radio-enhancement and cell cycle distribution suggests that the cell cycle effect does not constitute the most important mechanism of interaction with ionizing radiation. Our study also indicated that in the two cell lines studied, a modulation of the G1–S checkpoint was not implicated in enhancement of radiation response by dFdC.