Laboratorio práctico de poderes otorgados en el extranjero

Both SW837 and HRA-19 cell lines received a total dose of 48Gy (section 5.2.5), mimicking as closely as possible the long-course dosing regimen given to patients with rectal cancer within the clinic (section 5.2.5). The DRC results of SW837 and HRA-19 (Figure 29) suggested that a fractionated dose of 8 Gy may be suitable for both cell lines. A dose of 8 Gy was both clinically relevant and was enough to achieve significant cell death without killing all of the cell population (see raw colony counts in Appendix E-H) hence enabling the surviving cells to continue growing ready for the next dose. However, whilst the SW837 cell line appeared to cope well with the selected dosing regimen, as indicated by a sufficient number of surviving cells, it became increasingly apparent during the course of

0.001 0.01 0.1 1 0 2 4 6 8 10 S u rvival F rac tion

Radiation Dose (Gy)

SW837 HRA-19

119 the experiment that continued exposure to 8Gy fractions was not suitable for HRA-19, due to significantly increased levels of cell death between fractions. Therefore, due to the decreased number of cells surviving each irradiation dose, the decision was made to reduce each treatment fraction to fortnightly doses of 4 Gy (to a total of 48 Gy) for the HRA-19 cell line.

5.3.3 Confirmation of radioresistance

Figure 30 and Figure 31 show the experimental layout of one of the two modified colony counting assays set up for SW837 and SW837 (48 Gy), and HRA-19 and HRA-19 (48 Gy), respectively. It can be clearly seen that the SW837 cell line, and the novel derivative, show increased resistance to the effects of radiotherapy when compared to the HRA-19 cell line as distinguished from the greater number of colonies formed at each dose (see also Appendix E-H). As expected the greatest number of cells survived when exposed to no radiation in both cell lines. Both figures clearly illustrate that an increase in radiation dose lead to a decrease in subsequent colony formation.

120

Figure 30: Experimental layout of a modified colony counting assay for SW837 and SW837 (48 Gy).

Both SW837 and SW837 (48 Gy) were irradiated with a range of doses (0-10 Gy). One thousand cells from each dose were then seeded into 6 well tissue culture plates. Colonies that formed after 12-14 days were fixed, stained and counted in order to calculate the survival fractions. Each dose was plated in triplicate and each experiment was duplicated for confirmatory purposes. Both modified colony counting assays for SW837 and SW837 (48 Gy) were carried out at the same time, as 1 experiment. It was observed that the cell population at 0 Gy for the SW837 (48 Gy) formed much larger colonies than SW837 at 0 Gy, a difference possibly owing to changes in proliferation rate of the novel derivative.

121

Figure 31: Experimental layout of a modified colony counting assay for HRA-19 and HRA-19 (48 Gy).

Both HRA-19 and HRA-19 (48 Gy) were irradiated with a range of doses (0-10 Gy). One thousand cells from each dose were then seeded into 6 well tissue culture plates. Colonies that formed after 12-14 days were fixed, stained and counted in order to calculate the survival fractions. Each dose was plated in triplicate and each experiment was duplicated for confirmatory purposes. Both modified colony counting assays for HRA-19 and HRA-19 (48 Gy) were carried out at the same time, as 1 experiment.

122 Figure 32 shows the final log-linear survival curves for SW837 as compared with its novel RR (48 Gy) derivative, hereafter termed SW837RR, as determined by a modified colony counting assay. SW837 RR was significantly more radioresistant than its parental counterpart at 4, 6, 8 and 10 Gy (p≤ 0.05; Students t-test) giving a maximal 31-fold increase in resistance at 10 Gy Table 10.

Figure 32: The final log-linear curve illustrating the difference in radiosensitivity between SW837 and SW837 RR.

Each point represents the mean number of surviving cells from 6 replicates across 2 independent experiments. Compared to its parental corresponding cell line, SW837 RR (48 Gy) demonstrated a significant increase in survival at 4, 6, 8 and 10 Gy (p≤ 0.05; Students

t-test) with a maximal 31-fold increase in resistance observed at 10 Gy.

Table 10: Statistical confirmation of radioresistance for SW837RR compared with SW837.

Statistical analysis using the Students t-test confirmed significant radioresistance (p≤ 0.05) between the novel derivative (SW837RR) and its corresponding parental cell line (SW837) at doses of 4, 6, 8 and 10 Gy (shaded). A maximal 31-fold increase in resistance was observed at 10 Gy. Dose 0 Gy 2 Gy 4 Gy 6 Gy 8 Gy 10Gy p-value 1 0.978 0.020 0.001 0.000 2.92x10-5 Fold change 1 1 1.6 2 4.9 31 0.001 0.01 0.1 1 0 2 4 6 8 10 S u rvi va l F ract ion

Radiation Dose (Gy)

SW837 SW837 RR

123 Figure 33 shows the final log-linear survival curves for HRA-19 as compared with its novel RR (48 Gy) derivative, hereafter termed HRA-19 RR, as determined by a modified colony counting assay. HRA-19 RR was significantly more resistant than its parental counterpart at 4, 6 and 8 Gy (p≤ 0.05; Students t-test). A 4.2-fold increase in resistance was observed at 8 Gy (Table 11).

Figure 33: The final log-linear curve illustrating the difference in radiosensitivity between HRA-19 and HRA-19 RR as determined by a modified colony counting assay.

Each of the points represents the mean of 6 replicates from 2 independent experiments. Compared to its parental corresponding cell line, HRA-19 RR (48 Gy) demonstrated a significant increase in survival at doses of 4, 6 and 8 Gy (p≤ 0.05; Students t-test). At 8 Gy, a 4.2 fold increase in resistance was observed.

Table 11: Statistical confirmation of radioresistance for HRA-19RR compared with HRA-19.

Statistical analysis using the Students t-test confirmed significant radioresistance (p≤ 0.05) between the novel derivative (HRA-19RR) and its corresponding parental cell line (HRA- 19) at doses of 4, 6 and 8 Gy (shaded). A 4.2-fold increase in resistance was observed at 8 Gy. Dose 0 Gy 2 Gy 4 Gy 6 Gy 8 Gy 10Gy p-value 1 0.074 0.000 0.003 0.001 0.059 Fold change 1 1.2 2.8 3.5 4.2 59 0.001 0.01 0.1 1 0 2 4 6 8 10 S u r v iv al F r ac ti o n

Radiation Dose (Gy)

HRA-19 HRA-19 RR

124