Data analysis

There are various ways of expressing data obtained using PFGE (Ruiz de Almodovar 1994, Cedervall et a l 1995). The majority of studies on normal cells have measured the fraction of activity released (FAR: §2.7) (Blocher et a l 1989). This method is used as a measure of DNA DSB induction and repair. The measure of DNA is indirect and uses the distribution of radiolabel such as (incorporated into proliferating cells over a period of 96 h prior to the experiment in the present study) or fluorescence emission from fluorescent dye visualised by an image processor, as an actual measure of the DNA. The signal in the well is compared with that in the gel lane and this is taken as a measure of the amount of DNA that has migrated into the gel. The test lane can be corrected for the background level of damage by subtracting the amount of migration seen in the 0 Gy control well and DNA damage is expressed as %FAR where:

This provides an indirect measure of DNA damage and a measure of the average level of damage within the fragment sizes that are being separated. The FAR is dependent on the running conditions used, which particularly affect the threshold level of the DNA that is able to migrate into the lane. The FAR is not a linear response throughout the gel because of limits set by the electrophoretic running conditions. Compression zones, whereby DNA of a range of sizes remains in a bulk mass, and size inversion where larger DNA migrates further than smaller DNA can occur. Therefore it is not appropriate to use the fraction migrated into the gel as a measurement of DNA damage from gel to gel unless precisely the same running conditions are used.

In the present study, the dose-response curves for DNA damage were fitted by linear regression for each data set with the fitted line forced through the origin. An additional analysis was carried out to assess whether the y-axis intercept of the line would be significantly different from the origin if not forced through it. An analysis of variance (ANOVA) was used to compare the degree of intra-strain and inter-individual variation for both initial and residual DNA damage. The comparison between cellular radiosensitivity and DNA damage slopes was performed by linear regression analysis. Residual DNA damage was also expressed as a single dose point. The FAR at 70 Gy, the highest dose given, was chosen to maximise the difference between cell lines. Experiments were carried out a minimum of three times, with three plugs at each dose point.

The number of disintegrations per cell (DPM) was calculated to ensure adequate incorporation of the radiolabel (Table 5.4). The cell-cycle distribution of each cell line at

the time of irradiation was determined by propidium iodide incorporation (§2 .8).

Analysis was performed using flow cytometry and quantified using the Lysys II software program (§6.7.1: Becton Dickinson). Results are shown in Table 5.3.

5.5 R esults

Figure 5.1 shows a representative example of a 0.8% agarose gel stained with ethidium bromide following 48 h PFGE for the assessment of initial DNA damage. Each dose point is performed in triplicate and it can be seen that there is a good separation of DNA fragment sizes. As the radiation dose given is increased from 2-30 Gy, there is a corresponding increase in the 'leakage' of DNA from the wells. The molecular markers can be seen divided into bands, indicating that the running conditions were satisfactory for this gel. A similar result was seen for residual damage, although the amount of DNA 'leakage' is less following the 4 h repair period.

Results obtained with propidium iodide staining show that the percentage of cells in G 1 was >90% in all 18 primary cell strains (Table 5.3). A representative flow cytometric plot showing the regions set to calculate proportions of cells in each cell-cycle phase is shown in Figure 5.2. Although there was inter-strain variability in the proportion of cells in either S phase or G2/M phase, there was no correlation with HDR SF2 (G1 phase:

r=0.37, p<0.13; S phase: r=0.30, p<0.22; G2/M phase: r=0.47, p<0.05).

The amount of background DNA released from untreated 0 Gy samples ranged from 4.8 to 8.4% among the 18 fibroblast strains (Table 5.4). There was no correlation between HDR SF2 and background damage (r=0.02, p<0.91) or the number of DPMcell‘1 (Table

5.4: r=0.30, p<0.22).

An analysis of variance revealed significantly greater inter-individual heterogeneity compared with intra-individual differences in both initial (p<0.009) and residual DNA damage (p<0.0 0 0 1) in the 18 primary fibroblast strains.

Considering the linear fit for both initial and residual DNA damage graphs, in the majority of the non-syndromic strains the y-axis intercept was not significantly different from zero and so a linear fit through the origin was justified. For those strains where the intercept was significantly higher or lower than the origin, there was no correlation with radiosensitivity.

5.5.1 Initial DNA damage

Considering DNA damage induction, over the dose range studied (2-30 Gy) a linear relationship was seen between FAR and radiation dose for aU 18 primary fibroblast

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Figure 5.1. A photograph of a 0.8% agarose gel stained with ethidium bromide, destained and photographed on a transiluminator. The gel was subject to 48 h pulsed-field gel electrophoresis on a CHEF III machine (BioRad). As the radiation dose is increased from 2-30 Gy, the amount of initial DNA damage is increased as seen in a greater ‘leakage’ of DNA from the wells. Following electrophoresis, the well is removed from the lane and the amount of DNA in the gel quantified by scintillation counting. DNA markers are shown on the extreme right of the gel.

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