Manejo y actividad de enzimas y extractos enzimáticos

The retroviral vector used to infect cells with TP DNA was linked to a puromycin

resistance gene to allow selection. Therefore prior to these infection experiments,

2.3.1 Puromycin killing curves

Cells were routinely passaged and plated into medium supplemented from a puromycin

stock solution (5mg/ml) at concentrations ranging from 0 - 5 pg/ml. This medium was

changed every 3 days and the lowest concentration that caused 100 % cell death (usually

achieved within 7 - 1 0 days) was the killing concentration for that cell line.

2.3.2 Cell infection with retroviral vector containing TP DNA

The retroviral vectors used were a kind gift o f Mr Charlie Chan and Dr Rhys Jagger

(ICRF Oxford). Two vectors were used, AM 12 TP 12 which had previously been

shown to produce high expression o f TP when infected into cells, and puro 1, an em pty

vector containing only the gene for puromycin resistance.

Cells were passaged routinely at day 0. At 24 hours the medium was aspirated and

replaced with serum free medium (containing glutamine and antibiotics) and 1 0 pi o f virus,

(an amount previously shown to produce 1 0^ colony forming units per 1 0^ cells) was

added. In addition a 1/100 volume polybrene solution (800 pg/ml - Sigma) was also added

up as a control, as above but with the omission o f any virus. After 4 hours this medium

was aspirated into virkon, and replaced with routine complete culture medium.

When the plate reached confluence it was passaged in the normal way but this time the

medium was supplemented with puromycin at the killing concentration appropriate to

that cell line instead o f the routine antibiotics. Cells were refed every 3 - 4 days. After 7 -

10 days all the cells in the mock infection group were dead. Viable cells in the true

infection groups were allowed to grow to confluence, whereupon they were passaged and

seeded at a density o f only 1000 cells per 10 cm plate. This resulted in individual clones

growing up. When clones were approximately 100 cells in size they were 'picked'.

2.3.3 Picking of infected clones

Plates with clones o f appropriate size were washed in PBSA. A thin film o f PBSA was

left over these cells and individual clones were harvested by pipetting 2 0 |il o f trypsin-

EDTA up and down onto them. Each clone was then transferred to an individual well o f a

24 well plate and with growth gradually transferred to progressively larger plates, until

enough cells were obtained for continued passage, freezing down o f stocks and analysis o f

2.3.4 Plating efficiency experiments

At routine passage 1000 cells o f each cell line were seeded onto separate 10 cm plates.

When individual colonies were large enough to be seen by the naked eye but not so large

as to merge with other colonies the experiment was stopped, typically around 14 days.

At this stage the medium was aspirated and cells fixed with Cam oy's fixative (3:1

Methanol : Acetic acid) and stained with Crystal violet 1 mg/ml (Sigma). Colonies were

counted on an automated colony counter using 'sorcerer' software (Microsoft, USA).

2.3.5 Doubling times

Five X 10^ RT112, E V ll or 2T10 cells were seeded into 10 cm plates. Cells were

trypsinised and counted at regular intervals until confluence was reached, typically around

5 - 6 days. The doubling time o f each cell line was calculated during the exponential

growth phase.

2.3.6 W estern blotting for TP expression in infectant cells

To determine TP expression in the infectant cell lines, western blotting was performed,

according to section 2.1.11, with the following exceptions. Following harvesting, cells

were homogenised in Hepes buffer rather than 8M urea buffer (20mM Hepes pH 7.4, 1.5

inhibitor). Proteins were separated in 12% polyacrylamide gels (not 6%). The primary

antibody for TP was P-GF 44.C at a dilution o f 1 : 200, otherwise the procedure was the

same.

2.3.7 Immunohistochemistry for TP in the in vitro model see section 2.2.4

2.3.8 Effect o f TP overexpression on tumour growth in xenografts

This work was carried out with the help o f Gill Hutchinson and Julie Bee in the animal

house at ICRF, Lincoln's Inn Fields, London.

1 X 10^ cells, either E V ll (empty vector) or 2T10 (high TP expressing clone) were

injected into the flanks o f nu/nu mice. After injections mice were examined regularly to

confirm health and assess tumour formation. Perpendicular diameters o f palpable tumour

nodules were measured every 3 - 4 days with vernier callipers. Tumour volume in mm^

was calculated according to the formula;

Following sacrifice the tumours were snap frozen for subsequent western blotting for TP

(section 2.1.11 and 2.3.6).

2.3.9 Assessment of toxicity and effect of Furtulon

Furtulon (5-deoxy-5-flourouridine)(5-DFUR) is a prodrug which is converted to the

active metabolite 5-fluorouracil (5-FU) by the activity o f TP.

To investigate whether we could turn the high expression o f TP in our cell transductant

(and by extrapolation, possibly in invasive bladder cancers with their high expression o f

TP) to our advantage, we tested the effect of Furtulon on the in vitro model. Initially

M TT assays were performed to assess toxicity in vitro. Following that the chosen

concentration o f Furtulon was added daily to in vitro model cultures in 0.1 ml H2O similar

to other inhibitors (section 2.2.5).