Análisis de torque y arrastre

Many oncogenes identified as possible prognostic markers of tumour susceptibility and/or treatment response have been strongly implicated in the biological pathways examined in this study. Important cell cycle regulators such as p53 and cyclin B1 (Bristow, 1996; Maity et al. 1994) have been positively correlated with radiation response in many tumour cell types. Bcl-2, a key protein in the regulation of radiation

induced apoptosis has also shown prognostic significance in some tumour types (Wilson et al. 1996). Therefore, it was decided to analyse the expression of these proteins both constitutively and after radiation in order to check for any correlation with survival, and to identify any disorders in these pathways which may be caused by a mutated gene or inactive protein.

Protein expression in all cell lines was analysed by flow cytometry and/or western blotting. Most of the staining protocols for each individual protein varied only in the concentration of the primary antibodies and the fixative used. For this reason, the general staining procedure will be described here and any variations noted in the relevant chapter.

2.6.1 Flow cytometry 2.6.1.1 Sample staining

1. 1 X 10^ cells previously fixed in 70% ethanol, were washed twice in 5 ml PBS, at 2000 rpm for 5 minutes.

2. The supernatant was removed by aspiration and the pellet resuspended in 100 pL PNT containing a 1/25 dilution of the primary antibody, e.g. for p53, 4 pL of mouse anti-human p53 (DO-7, Dako) was added to 96 pL PNT.

3. Incubation was at room temperature for 1 hour.

4. Cells were washed in 5 ml PBS and the pellet was resuspended in 100 pi PNT containing a 1/25 dilution of a secondary goat anti-mouse FITC conjugated antibody (Sigma).

6. Incubation was at room temperature for 1 hour, in darkness.

7. Cells were washed in PBS and resuspended in 1 ml of PBS containing 1 mg/ml RNAse and 20 pg/ml PI.

Positive controls were prepared by the same protocol. Normal fibroblasts were used as standards for all the proteins analysed. Samples stained with an isotypic antibody instead of the primary antibody were used a negative controls. A sample known to be negative for the protein, e.g. HL60, which are null for p53 protein expression, were also used as negative controls where possible.

2.6.1.2 Sample collection and analysis

All samples were collected and analysed on a FACScan flow cytometer, using the LYSIS II analysis software (Becton Dickinson). Channel settings, FLl (protein) and FL3 (DNA), were determined on the negative and positive control samples, with FLl emission set on a linear scale. Samples were analysed using computer generated regions set on the negative control samples to contain <1 % of the total population. This accounts for any non-specific binding effects of the antibodies on the overall positivity.

This region was then overlaid onto the test samples and the positivity calculated by the number of cells in this region as fraction of the total.

2.6.2 W estern blotting 2.6.2.1 Harvest and fixation

Samples were harvested by scraping and were washed twice with cold PBS. The pellet was resuspended in 1 ml PBS and transferred to an eppendorf tube. Samples were centrifuged for 5 minutes at 2000 rpm. The supernatant was aspirated and the pellet stored at -70 °C until use.

2.6.2.2 Sample preparation and loading

Cells were resuspended in 100 \iL loading buffer (see Appendix 1), sonicated for 5-10 seconds at 30 mA (Ultrasonics Ltd.), and placed on ice. Samples were loaded on a 12% polyacrylamide gel (see Appendix 1) at a fixed cell number of 0-5 X 10^. Rainbow (14.5-220 kDa) and ECL (Amersham International) protein markers were run at each end of the gel.

2.6.2.3 Running protocol and protein transfer

All samples were run first through a layer of 12% polyacrylamide stacking gel (see Appendix 1), to align the samples, and then through a 12% polyacrylamide running gel (see Appendix 1). Running conditions were 50 V and 150 mA, at room temperature for eight hours, in running buffer (Appendix 1). Samples were then transferred to a Hybond-ECL nitrocellulose membrane (Amersham International) in transfer buffer (see Appendix 1) overnight at 4 °C, with an applied voltage of 50 V and 200 mA.

2.6.2.4 Membrane staining

1. Visualisation of the proteins was achieved using a Ponceau stain (Sigma) for 10 minutes.

2. The membrane was then washed in 5% acetic acid ( 3 x 5 minutes), air dried and photocopied for a visual record of the gel.

3. Non-specific binding sites are blocked by incubation for 1 hour in blocking buffer (see Appendix 1) containing 5% non-fat dry milk (Marvel).

4. Primary antibody solutions were made up in blocking buffer containing 1% non-fat dry milk. Incubation was at room temperature for one hour, with slow agitation.

5. The membrane was washed for 3 X 10 minutes with wash buffer (see Appendix 1). 6. The secondary antibody was a goat anti-mouse horseradish peroxidase-conjugated antibody, which was diluted to a standard concentration of 1/10 000 in blocking buffer containing 1% non-fat dry milk.

7. A secondary antibody for the ECL protein marker was also added, at a concentration of 1/1500. Incubation was at room temperature for 45 minutes with slow agitation. 8. The membrane was then washed for 3 X 10 minutes in wash buffer (see Appendix 1).

2.6.2.5 Protein visualisation using enzyme chemiluminescence (ECL)

This was carried out as instructed in the ECL kit manual (Amersham International). 1. A solution consisting of equal volumes of detection reagents one and two was added to the protein side of the membrane. Incubation was for 1 minute without agitation. 2. The membrane was washed by holding vertically over tissue paper, gently touching the edge.

3. The membrane was then placed, protein side down on some cling film and covered. 4. Once wrapped the membrane was placed, protein side up in a film cassette, taking care to ensure that any air bubbles were removed.

5. The membrane was then exposed autoradiograph film, (Hyperfilm-ECL, Amersham International) in a dark room for -15 seconds, depending on the intensity of the signal. 6. The autoradiograph was developed for 3 minutes and fixed.