The role of apoptotic cell death in the radiosensitising effect of gemcitabine.

BACKGROUND: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis. METHODS: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment. RESULTS: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment. CONCLUSION: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

Feasibility of collecting self-sampled vaginal swabs by mail: quantity and quality of genomic DNA.

Vaginal self-sampling may be valuable as an alternative method of cervical cancer screening in areas of poor resources, to enroll women who, otherwise, would not participate in population-based cervical cancer screening and in epidemiological follow-up studies. We assessed the reliability of mailed vaginal samples by evaluating the quantity and quality of genomic DNA in the samples. Mailed swabs (n = 201) were compared with freshly collected samples (n = 200) for DNA concentration (45.1 versus 50.9 ng/microl, respectively) and purity (mean optical density [OD] 260/280 ratio 1.88 versus 1.78, respectively). A small, non-significant, decrease in DNA yield with longer transport time was noted. The DNA yield of mailed samples was significantly lower compared to fresh samples (P < 0.002), but this lower yield had little effect on polymerase chain reaction (PCR) amplification. In conclusion, the large majority of mailed self-sampled vaginal swabs resulted in DNA of adequate purity and concentration for further research.

In vitro interaction between ecteinascidin 743 (ET-743) and radiation, in relation to its cell cycle effects.

Ecteinascidin 743 (ET-743) is a new marine-derived agent with promising activity against a number of solid tumours. In four human tumour cell lines, the interaction between ET-743 and radiation was investigated in relation to the effects of ET-743 on the cell cycle, in vitro. Cell survival was measured based on quantitative staining of cellular protein by sulforhodamine B. A 24 h treatment with ET-743 before radiation resulted in a moderate increase in radiosensitivity in three out of four cell lines. Dose enhancement factors > or =1.8 were observed for concentrations resulting in 52, 46 and 30% cell kill in ECV304, H292 and CAL-27, respectively, whereas in A549 no radiosensitisation was observed (no significant increase in radiosensitivity). According to the combination index analysis, synergism was observed only in ECV304 and CAL-27 cells. A 24 h incubation with ET-743 resulted in a concentration-dependent G2/M block, which might explain the moderate radiosensitising effects in ECV304 and H292. The lack of radiosensitisation in A549 might be due to the S phase delay preceding the G2/M block at the moment of radiation, which only occurred in this cell line. In conclusion, ET-743 has moderate cell line-dependent radiosensitising properties; however, only when cytotoxic concentrations of ET-743 are used. In one of the four cell lines tested, no radiosensitisation was observed.

The radiosensitising effect of gemcitabine and the influence of the rescue agent amifostine in vitro.

In this study, the radiosensitising effect of different concentrations of gemcitabine and the combination of gemcitabine/radiotherapy with the rescue agent amifostine was investigated in different human tumour cell lines. The cells were treated with gemcitabine (0-8 nM) for 24 h prior to radiation (0-8 Gy). Amifostine (ami) and alkaline phosphatase (AP) were added 30 min before radiation. Cell survival was determined 7 or 8 days after radiation treatment by the sulforhodamine B (SRB) test. For ECV304 cells, the dose enhancement factor (DEF) varied from 1.39 to 2.98 after treatment with 1-6 nM gemcitabine. FaDu, H292, A549 and CAL-27 seemed to be less sensitive, with DEFs ranging from 1.02 to 2.67. These cells were also less sensitive to the cytotoxic effects of single-agent gemcitabine. Amifostine with AP clearly showed a protective effect in combination with gemcitabine/radiotherapy. In H292 cells, the protection factor (PF) of amifostine after treatment with gemcitabine and radiotherapy varied from 1.64 to 1.86. In ECV304 cells, the PF varied from 2.20 to 2.29. In conclusion, a clear concentration- and cell line-dependent radiosensitising effect of gemcitabine was observed in all cell lines. Amifostine with AP showed protection against the radiosensitising effect of gemcitabine. If the protection in vivo indeed occurs selectively in normal tissues, then amifostine could prevent or strongly minimise the increased toxicity resulting from the radiosensitising effect of the combination of gemcitabine and radiotherapy, without influencing the antitumour effect.