DEFINICIÓN DE LA MINERÍA DE DATOS

Maintenance of Cell Cultures

All cells used were obtained from the ICRF central cell services. Lymphoblastoid cell lines were maintained in 2% RPMI 1640 cell culture medium supplemented with 10% foetal calf serum (Gibco BRL). Cells were maintained at a concentration o f between 2x10^ and 2x10^ cells/ml at 37°C in a humidified atmosphere containing 5%

CO2.

Fibroblast lines, colorectal lines and the variants o f A2780 were m aintained in Dulbecco's MEM (E4) supplemented with 10% FCS at 37°C and 10% CO2. Cells

were passaged approximately twice a week by detaching the cells with a thin layer o f 0.25% Trypsin in 0.1% Versene for 3 to 5 minutes at 37°C. This was neutralised with 10 ml o f medium containing 10% FCS and cells replated at dilutions o f between

1:5 and 1:12.

Large scale cell culture for cell extracts was perform ed by the ICRF central cell services laboratory according to their standard methods.

Viable cell numbers were counted using an Improved Neubauer haemocytometer. At least four large squares were counted, the number averaged and multiplied by 1 0'^ to

give the number o f viable cells/ml.

Cell Storage

Frozen cell stocks w ere stored at -70°C and under liquid nitrogen (LN2).

Approximately 2 to 5x10^ cells were pelleted, the medium removed and the pellet resuspended in the appropriate growth medium containing 10% (v/v) DMSO. Cell suspensions were transferred to cryovials (Nunc), wrapped in tissue paper, placed at - 70°C and cooled at a rate o f 1°C / minute. After 48 hours, vials were transferred to LN2 for long term storage.

Cell stocks were recovered by rapidly thawing the cryovial at 37°C and transferring the cells into an appropriate volume o f fresh media.

Generation of Cell Cultures From Single Cells

Single cell clones were established by limiting dilution. Essentially, an exponentially growing cell culture was diluted to 20, 40, 60 or 80 cells per ml. lOOpl o f diluted cells were seeded in each well o f a 96 well plate and allowed to grow for 2-3 weeks. Mass cultures were established from wells containing only a single colony.

Selection of Cisplatin Resistant Clones

A medium flask containing an exponentially growing, sub-confluent population o f approxim ately 5x10^ A 2780-SC A 5 cells w as treated w ith an appropriate concentration o f cisplatin (3.33mM Stock in 1% NaCl, David Bull Laboratories) in growth medium containing 10% FCS. Treatment was for one hour and after this period, the medium was replaced with drug-free medium containing 10% FCS. Cells were then left to recover for about 2-3 weeks before the next treatment. A fter the cells had recovered from the final treatment, single cell clones were generated.

Dialysis of Foetal Calf Serum

Foetal calf serum was dialysed against 10 x volume o f PBSA using SpectraPor? dialysis tubing (Fisher Scientific) at 4°C for 48 hours with five changes o f PBSA before being filter sterilised. The dialysis tubing has a molecular weight exclusion limit o f 2KDa.

Cell Survival by Colony Formation Assay

Cisplatin

Cells from an exponentially growing culture were seeded at between 200 and 10^ per 10cm dish and allowed to attach for about 4 hours. A n appropriate concentration o f cisplatin was added and cells were incubated for a further hour. The growth medium was then replaced with fresh medium, the cells incubated for a further 1 0 days and

S'TdR and UVA

Cells were grown for 3 days in medium supplem ented w ith 10% DFCS and the indicated concentration o f S^^TdR. Cells were counted, serially diluted and seeded at between 100 and 10^ per well o f a 6 well plate. Cells were allowed to attach for 4

hours and then washed once with 5mis o f warm PBSA before being irradiated with either a V L-6L (Merck) or a UVH-253 (UV Light Technology, Birmingham, UK)

lamp under a thin film (approx. 1mm) o f PBSA. The dose rate was calibrated using either a J-221 (UVP) or UV-A (UV Light Technology) meter. After irradiation, the PBSA was replaced with fresh growth medium containing 10% FCS and viable colonies scored after a ftirther 10 days growth by Giemsa staining.

and UVA

Experiments were carried out as above except cells were grown in undialysed FCS.

Lymphoblastoid Growth Curves

Cells from exponentially growing cell stocks were seeded at a concentration o f 5x10^ cells per ml in a 24 well plate and grown in the continuous presence o f the drug. S^G was dissolved in 0.1 M NaOH as a 5mM stock and S"*TdR as a 20mM stock in dH2 0 .

Cell numbers were determined by daily cell counts using an improved Neubauer haemocytometer.

UVA Irradiation

Lym phoblasts were grown in medium containing 10% DFCS and the appropriate concentration o f S^^TdR for 3 days. Cells were then washed once w ith PBSA and irradiated as before as a concentrated suspension (~l-5xlO^ cells/ml) in PBSA. Cells were pelleted and resuspended in fresh growth medium containing 10% FCS and seeded at a concentration o f 5x10^ cells per ml in a 24 well plate. Growth was then determined as above.

Mutation Frequency

M utation frequency was determined at the A P R T locus using the CHO cell line D422 (G onclaves et al., 1984). Cells were grow n for 3 days in grow th m edium

supplemented w ith 10% DFCS and lOOpM S^^TdR. After this period, cells were expanded for a further 5 days in growth medium containing 10% FCS and no S"*TdR. Cells were counted and plated at a concentration o f 5x10^ per 10cm dish. A P R T mutants were selected using 0.4mM 8-azaadenine and the num ber o f mutants per

plate determined after a further 10 days growth. At least 1.5x10^ cells were plated per experiment.

Inhibition of Cell Growth by Aminopterin

500pl o f Raji or Jurkat cells grown in either 10% FCS or 10% DFCS were seeded at a concentration o f 4x10^ per ml in 24 well plates before being treated with various combinations o f 0.4pM aminopterin (GibcoBRL), lOOpM hypoxanthine and various concentrations o f TdR or S^^TdR. Cell numbers were determined after a further 3 days growth.