Revisión Periódica

3.3.1 Introduction

One of the aims of this thesis is to test whether a similar phenomenon to HRS occurs after low dose-rate exposures. This chapter describes how cell lines were tested for the

presence of HRS at low-acute doses. Chapter 4 describes the results of some of these cell lines after low dose-rate irradiation.

So far 25 cell lines have been tested for the presence of HRS/IRR; of these 5 have not shown the effect (Table 1.4). Two of these, the SW48 human colonic carcinoma and HX142 human neuroblastoma, have SFjS of below 0.2 (SW48 0.179 and HX142 0.033) and this is extremely radiosensitive compared with the majority of other cell lines tested. The two other cell lines, that do not show HRS/IRR are relatively radioresistant (SF2S of

0.62 U373MG and 0.63 SiHa). These are useful cell lines to study in comparison with HRS+ve lines as differences may elucidate possible mechanisms of HRS/IRR. For example SW48 is mismatch-repair deficient, suggesting that this may be involved in HRS/IRR. When the a /a ^ values are plotted against SFj for all 25 cell lines (Figure 3.14), there is a general trend that the more radioresistant a cell line, the greater the degree of HRS observed, producing a positive trend with r= 0.199, although this was not

significant, p= 0.333. This phenomenon therefore appears to be more characteristic of radioresistant cell lines. It is possible that the repair mechanisms involved in IRR in these cell lines also contribute to resistance seen at higher doses. This may explain why the cell lines with very low SF^, SW48 and HX142, do not show HRS as they may have an absence of these repair mechanisms and are therefore radiosensitive along the whole survival curve.

Of the 25 cell lines studied, 8 have been assessed using the DMIPS protocol and 14 using

the cell sorter. 3 cell lines have been tested using both methods. Short (1999b) studied HRS in a panel of human glioma cell lines with wide ranging radiosensitivity. They tested two of the cell lines used in this thesis, T98G and U373MG, using both protocols. As HRS/IRR was not present in U373MG no IR fit was obtained for comparison between protocols. The SF^ was very similar using both protocols (0.62 DMIPS and 0.65 cell sort). Comparing the results from the two protocols tested on asynchronously growing T98G, the cell sorter gives a slightly higher SFj value (0.73 compared with 0.62). The degree of HRS/IRR is very similar for both protocols with the a /a ^ value being 13.2 for the sorter and 14.3 for the DMIPS. These results suggest that the two methods are sensitive enough to detect the presence, or in the case of U373MG, the absence of

HRS/IRR. The human colon adenocarcinoma cell line, HT29, has also been studied in two laboratories using the two protocols (Lambin et a l 1993, Wouters et a l 1996). The

results were consistent as they both indicated the presence of HRS/IRR, although a larger

aja^ value was obtained for the DMIPS data, 20.54 (Lambin et al. 1993) compared with the cell-sort protocol, 6.89 (Wouters et al. 1996).

3.3.2 Assessment of acute dose cell survival

Two methods were employed to accurately assess cell survival at very low-acute doses. They are described in detail in Section 2.6.

3.3.2.1 Dynamic Microscopic Image Processing Scanner

The DMIPS was used to obtain accurate survival curves (Section 2.6.1) for the following cell lines: RTl 12, T98G (confluent), A7, LNCaP, PC3 and DU145. A photograph of the DMIPS is shown in Figure 2.4.

Confluent T98G cells were grown as described in Section 2.4.1.3 and were used in these experiments only after they had been observed as confluent for 4 days.

During a single experiment 3000-3500 cells were plated into 16 flasks. This cell number gives a maximum number of logged locations in the C-scan program without allowing overlap of colonies when they reach >50 cells (see Section 2.6.1). 16 flasks are the maximum number that can be handled by this protocol in one day. Flasks were incubated with their lids loose with 5% CO2 + 5% O2 (balance Nj) for 40-60 min to allow the cells

to attach. Attachment time varied between cell line. Flasks were then filled with fresh medium. Of the 16 flasks plated, 3 were 0 Gy controls which were sham irradiated on the X-ray set for 2 min. The other 13 flasks were irradiated as described in Section 2.5.1 at doses between 0.05 Gy and 5 Gy, with 1 flask per dose. After 10-14 days incubation, flasks were S-scanned. The number of days incubated depended on the doubling time of the cells. At least 3 experiments were carried out per cell line.

3.3.2.2 FACSVantage cell sorter

The following cell lines were assessed for cell survival using the cell-sort protocol (Section 2.6.2): T98G (confluent), A7, LNCaP, PC3 and DU145.

As with the DMIPS protocol, confluent T98G cells were observed as confluent for 4 days (Section 2.4.1.3).

The cell sorter was used to dispense a known number of cells into a 25 cm^ flask containing 5 ml medium. The number of cells dispensed depended on the plating

efficiency of the cell line and the dose of radiation the cells would receive. Sufficient cells were plated to give 100-200 surviving colonies per flask. 3-6 flasks were plated per dose point. 6-12 of these flasks were designated controls. Flasks were incubated for 2 hours

prior to irradiation. 2 hours allowed enough time to for cells to attach, but not enough for

them to divide. Irradiations were carried out as described in Section 2.5.1 at doses between 0.05 Gy and 5 Gy. Flasks were then incubated for 10-14 days and then stained with crystal violet and colonies counted using a manual colony counter.

3.3.2.3 Data analysis

In DMIPS and cell-sort experiments the plating efficiency (PE) was calculated using the following equation:

PE =

number o f colonies