To increase the efficiency of combined treatments, particularly the combination of DTIC and protons, the order of administration of drugs and radiation was inversed. The new experimental set up was conceived knowing the position on the time scale where the best effect of each single treatment with FM, DTIC or protons was reached [10]. The HTB140 cells were irradiated with protons, incubated for 4 days, when FM or DTIC was added to the cells, and then incubated for another 3 days. In this way it was enabled that the incubation periods providing the best single effects of protons and

drugs coincide at the same time. The described combination of protons and FM reduced cell proliferation to ~40% and clonogenic survival to ~50%, while there was ~80% of viable cells estimated by the SRB assay (Figure 1). With respect to the single treatments the obtained effects were weaker. The Fludarabine concentration time interval between irradiation and drug treatment might be considered as long because the multiplicity of microcolonies 4 days after irradiation could underestimate the effects of drug treatment, particularly for the clonogenic assay. An overestimation of cell viability by the SRB assay could be ascribed to the excess of proteins coming from the dead cells that were indistinguishable from those of surviving cells [23]. DNA damaging agents also produce morphological changes of cells, such as an increased cell size and therefore

protein content [29]. This might also explain the overestimated viability obtained by the SRB assay. Comparing the inactivation levels obtained in this experiment to those of the two experiments that PRIMA-1MET were previously described [11, 12], the best effect was obtained when the HTB140 cells were treated with FM before proton irradiation and incubated for 7 days [12]. The combination of protons and DTIC reduced cell proliferation to ~32% while after single treatments this level was higher (Figure 2). Again, an overestimation of viability was obtained by SRB assay [23, 29]. According to cell proliferation

and survival, the poor efficiency of the single DTIC treatment was overcome when it was introduced following proton irradiation. The cells that were damaged by protons and would most likely survive were additionally damaged in a similar way by the DTIC treatment [30]. Rutecarpine As a result, the obtained cell inactivation levels were better than those of the two previously reported experiments [11, 12]. Analysing the effects of the two administration procedures of radiation and drugs, in general there was not an appreciable improvement with respect to the single treatments. In each of them there was a moderate improvement with the combination of just one drug and radiation. All studied agents affect cellular DNA, but they differ in the type of damage they induce. Protons, as well as conventional radiation, induce oxidative changes in DNA bases together with the single- and double-strand breaks [31].