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Chemicals were obtained from Gibco BRL Life Technologies, M erck/BDH , BioRad Laboratories, National Diagnostics, Anachem Ltd. and the Sigma Chemical C om pany unless otherwise stated. Recrystalized N-m ethyl-N-nitrosourea (MNU) was a kind gift of Prof Peter Swann (University College London, London, UK) and potassium diazoacetate (KDA) was a generous gift from Dr David Shuker (MRC Toxicology Unit, University of Leicester, UK)

Radiochemicals.

H ypoxanthine-8-l^C (49.5mCi/mmol) was purchased from Sigma (UK). Redivue [y 32P] dATP (500Ci/mmol) was purchased from Amersham International pic.

Cell lines.

All cell lines, including the SV40 transform ed fibroblasts GM0637 and XP12RO, the endom etrial tum our cell line HeLa MR, the ovarian carcinoma cell line A2780, the mouse monocyte macrophage J774A.1 and the lymphoblastoid cell lines GM2345 (XPA), GM2252 (XPB), GM2498 (XPC), GM2249 (XPC), GM2248

(XPC), GM2485 (XPD), GM2449 (XPV), were obtained from the Cell Production Laboratory Imperial Cancer Research Fund at Clare Hall. Raji Mex+ and Raji Mex" w ere originally obtained from the American Type C ulture Collection, Rockville, M aryland. RajiF12 and Raji RIO were established as a variant of Raji Mex" in this laboratory (Branch et al., 1993a), (Hampson et al., 1997).

Methods.

Standard Solutions Used Throughout Experimental Procedures.

Disodium ethylenediam inetetraacetic acid (EDTA), Tris EDTA, phosphate buffered saline (PBSA) and Tris borate EDTA solutions were prepared as described by (Sambrook et al., 1989) and diluted as required.

Maintenance of Cell Cultures.

All lym phoblastoid cell lines, as well as the m urine m acrophage cell line J774A.1, were m aintained at betw een 1x10^-2x10^ cell/m l at 37^C in 2% RPMl 1640 m edium (Gibco) supplem ented with 10% foetal calf serum in a hum idified atm osphere containing 5% CO2. All SV40 transform ed fibroblastoid cultures and tum our cell lines, were routinely m aintained at around 10^ cell/m l at 37^C in Dulbecco's modified Eagle's m edium supplem ented w ith 10% foetal calf serum. Stocks were stored at -80^C and in liquid nitrogen in 2ml of the appropriate grow th m edium supplem ented with 10% dim ethylsulphoxide in Nunc cryotubes. Cells were retrieved by thawing in a 37^C water bath and resuspended in 10-15ml of the appropriate growth medium.

Measurement of Protein Concentrations in Aqueous Solution.

Protein concentrations were estim ated according to (Bradford, 1976). An aliquot of extract (typically Ipl) was added to 900|il Bradford reagent and the volum e m ade up to 1ml w ith d H 2 0 . After thorough mixing the absorbance of 595nm wavelength light by the solution (A595) was determ ined and the A595 of a preparation w ithout extract subtracted. The concentration of protein in this sample was estim ated by means of a standard curve established by determ ining the A595 of samples w ith known amounts of Bovine Serum Albumin. Bradford Reagent is composed of

lOOmg Coomassie Brilliant Blue G-250 50ml 95% (v /v ) Ethanol, 100ml Orthophosphoric acid (85% (v/v))

Made up to 11 with d H 2 0 and stored at 4°C protected from light.

Measurement of Nucleic Acid Concentrations in Aqueous Solution.

Concentrations of DNA in solution were determ ined by the m ethod of (Sambrook et a l, 1989). The optical density of solutions was determ ined at 260 and 280nm (A26O and A28O) iri quartz cuvettes. 1 optical density at 260nm corresponds to approxim ately 50m g/m l dsDNA, 40m g/m l RNA and 20m g/m l single stranded oligonucleotide. The ratio A26O/A28O is an estimate of sample purity. Ratios of 1.8 to 2 are associated with pure samples.

Isolation of Genomic DNA from Cells.

Small scale DNA preparations of high purity were obtained by pelleting cells, washing twice in PBSA, resuspending the pellet in extraction buffer (50-100|il

extraction buffer per 10^ cells to be extracted; lOmM EDTA, 50mM Tris.HCl (pH 8.0), 0.5% w /v sodium-lauryl sarkosinate, 0.5 m g /m l Proteinase K) and incubating the lysate for 2 hrs at 50^C. After 1 hr of the incubation, lOpl RNAaseH (lOmg/ml) w ere added per 20pl extraction buffer used. After com pletion of the 2 hrs incubation, and equal volume of phenol:chloroform:isoamyl alcohol (ratio 25:24:1 (v /v )) was added. After brief vortexing, the sam ples were centrifuged at room tem perature to separate phases. The upper phase was processed by repeating the phenol:chloroform :isoam yl alcohol treatm ent. The top phase from the second phenol:chloroform:isoamyl alcohol treatm ent was treated by addition of an equal volume of chloroform, brief vortexing and removal of the top phase. To this 1 /3 volum e of 3M sodium acetate (pH 5.2) and 2 volumes of ethanol (-20®C) were added and the DNA allowed to precipitate for 1 hr at -70^C. Samples were pelleted using a benchtop Sigma centrifuge, the supernatant rem oved and the pellet resuspended in TE.

O^-Methylguanine-DNA methyltransferase (MGMT) Assay.

MGMT activity was determ ined using the protocol described by (Harris et

a/., 1983).

Cell Extracts for MGMT Assay.

Cell extracts were prepared by lysis of approximately 10^ cells in 100-200pl of Triton extraction buffer (50mM Tris.HCl (pH7.5); lOmM DTT; Im M EDTA; 0.2% (v /v ) Triton X - 100; stored at -20OC); lysate was clarified by centrifugation (5 m inutes at llOOOxg in a Sigma 113 Laboratory Centrifuge) and the protein concentration of the supernatant determined.

MGMT Assay: Substrate and Reaction Conditions.

M. leutus DNA w as m ethylated w ith N-[m ethyl-^H ]-N '-nitrosourea as

enzym e substrate; heat treatm ent of the DNA removes heat labile N-methylated bases thus increasing the p roportion of O ^m eG ua in the total am o u n t of m éthylation damage (Karran et ai, 1979). Enzyme reactions (10-100|ig extract) were perform ed in lOOpl assay buffer (70mM H epes KOH pH 7.8; lOmM DTT; Im M EDTA; substrate DNA so that approximately 1.5 x 10^ counts per lOOpl; stored at - 20^C). Reactions were incubated 20 m inutes at 37^C protein and substrate DNA precipitated by addition of 120pl ice cold 0.8M trichloroacetic acid and lOpl carrier DNA (2m g/m l heat denatured herring sperm DNA) and incubation on ice for 5 m inutes. The precipitate was pelleted by centrifugation at ISOOOxg for 10 minutes at 4 °C (Hereus Biofuge 17RS centrifuge) and the su p ern atan t rem oved and discarded. 0^-[^H]m eGua in DNA was hydrolysed by incubation w ith lOOpl of O.IM HCl. This does not hydrolyse [^H]methyl groups associated w ith the methyltransferase therefore, transferred and non transferred m ethyl groups can be distinguished. After incubation on ice for 5 minutes and centrifugation (1 minute, lOOOxg at room temperature) 80pl of the hydrolysate were added to 5ml Picofluor (Picofluor 15, Packard) and activity m easured in a liquid scintillation counter (Tricarb 1500 Liquid Scintillation Counter, Packard).

Calculation of MGMT Activity

In order to estimate the amount of radioactivity removed from the DNA by incubation w ith cell extracts, the count of radioactive disintegration's per minute (dpm ) in the sam ple containing extract is subtracted from the n u m ber of

d isintegration's detected in the control reaction w ithout protein. The specific activity of the substrate is approximately 5x10^ dpm per pmole DNA, the counting efficiency is assum ed to be 20% thus approximately 10^ counts per m inute (cpm) p er pm ole substrate will be recorded. If the data are plotted as am ount of radioactivity rem oved versus the am ount of protein used in the incubation, the slope of the graph represents the cpm removed per mg of protein incubated with. The MGMT activity of the extract can thus be calculated as:

slope X 10^ = cpm /m g

1Q4 cpm /pm ole

w ith 1 unit of methyltransferase being able to remove 1 pmole of O^meGua.

Hypoxanthine Phophoribosyl transferase (HPRT) Assay.

This assay is based on the form ation of inosine m onophosphate from hypoxanthine-8-l^C by cell extracts.

Cell extracts were prepared from 10^ cells using 100-200|il extraction buffer (lOmM Tris.HCL pH 7.4, lOmM MgCl2, 30mM KCl, O.lmM DTT, 0.5% Triton XlOO). Extracts (0-25mg) were incubated in 50|li1 reaction mix (50mM Tris.Cl pH7, 9mM MgCl2, 2mM DTT, 2mM phosphoribosyl pyrophosphate, [^^C] hypoxanthine at lOmCi/ml) for 15mins at 37^C, then transferred to 70^C for 5mins, before being p u t on ice for Smins. Triplicate lOpl aliquots of the mix were then spotted onto DE81 paper (Whatman), washed twice in 200ml 50mM Tris.Cl p H8, dried and counted in 5ml permablend. HPRT activity was calculated using the slope of the plot of counts per minute against |ig protein extract and expressed as

p m o l/m in /p g where one unit of enzyme activity liberates 1 p m o l/m in /p g .

Total G lutathione-S-transferase (7GST) and Glutathione-S-transferase-7C (7GST- 7c) Assays.

Total glutathione-S-transferase (7GST) and Glutathione-S-transferase-7C (tGST-tc) activity were determined spectrophotometrically by the m ethod of (Habig

et a l, 1974). Basically, cell extracts were prepared as for the MGMT assay and ^

incubated (0-200|Xg for total 7GST and O-lOOpg for 7GST-7C) in O.IM potassium phosphate (pH6.5) with reduced glutathione (2mM for total 7GST and 0.25mM for TGST-tc) and substrate (2mM l-Chloro-2,4 dinitrobenzene for total 7GST and 0.2mM Ethacrynic acid for 7GST-71) at 25®C in a final volume of Im ls for 30mins. Both substrates solutions were m ade up in water. For total 7GST, absorbance at 340nm with e=9.6mM'icm"i and for 7GST-71 absorbance at 270nm w ith e=5mM'icm‘ 1 was used (Habig et al., 1974). A plot of absorbance (at either 340nm was 270nm) against mg protein extracts was used to calculate enzyme activity which was expressed as n m o l/m in /m g .

D eterm ination of Acid Soluble Thiols.

Cells (5X10^) were sonicated in around 200fxl ice-cold PBSA and protein concentration determined using the crude sonicate. Specific amounts of sonicate (0|ig-200|L ig) were extracted in 200|iL 5% TCA. Acid insoluble material was pelleted and !00|li1 of the supernatant was added to 900|il of Ellmans reagent (0.2m g /m l of 5,5'-Dithio-bis(2-nitrobenzoic acid) in IM Hepes pH 7.8). The absorbance at 410nm was calibrated against a standard curve made using reduced glutathione as the acid soluble thiol. Levels of acid soluble thiols were expressed as fxmol.

y-Glutamyl Transpeptidase (y-GGT) Assay.

This assay monitors the y-GGT catalyzed transfer of the y-glutamyl moiety from the substrate L-y-glutamyl-p-nitroanilide to glycylglycine, thus releasing the reaction product p-nitroaniline, which has a maximum absorbance at 410nm. Extracts were prepared by sonicating cells (10^) in around 200|xl ice-cold PBSA and protein concentration was determined using the crude sonicate. Protein (0-500|ig) was incubated for 1 hr at 37^C with 2.5mM L-y-glutamyl-p-nitroanilide, 30mM glycylglycine (brought up to pH 8.5 with IM NaOH), 0.075M NaCl, 0.05M

Tris.HCl pH 8.8 in a volume of 1ml. The reaction was term inated by the addition of 20|xl glacial acetic acid and the tubes clarified by spinning in a benchtop centrifuge. The absorbance of the supernatant was determined at 410nm. p-Nitoroaniline purchased from sigma was found to have a £=6.8nM"l.cm“l. y-GGT activity was expressed as n m o l/m in /m g .

Greiss Reaction: an Indirect Determination of Nitric Oxide Concentrations.

This assay is used as an indirect determination of nitric oxide concentration the basis of which relies on the formation of nitrous acid under acidic conditions from nitrite (N 02“), a breakdown product of nitric oxide. Nitrosation of sulphanilic acid by nitrous acid in the presence of N-l-nathyl-ethylenediamine

dihydrochloride forms a diazo compound (purple in colour) which has a

maximum absorbance at 550nm. This assay was used to monitor the formation of nitric oxide derived nitrite from the activated murine macrophage cell line J774A.1. Basically, lOOpl of medium from the simulated macrophage culture was added to 900|il of reaction mix containing 0.5% (w /v) sulphanilic acid, 0.002% (w /v ) N-l-nathyl-ethylenediamine dihydrochloride, 14% (v/v) glacial acetic acid. Reactions were incubated at 37^C for 5-10 mins before reading the absorbance at

550nm. The formation of the purple diazo compound can only be m onitored using cell culture m edium without phenol red. Nitrite concentrations produced from the activated macrophage cultures were estimated from a standard curve using

sodium nitrite.

Levels of DNA Platination in Cultured Cells.

The levels of DNA platination in GM0637, XP12RO and MNU B4 were determined by atomic absorption (AA) spectroscopy by Dr Lloyd Kelland and Mr Ciaran O'Neil at the CRC Center for Cancer Therapeutics, Sutton, UK.

Approximately 4x10^ cells/cell line were treated using cisplatin (0, 25,50 lOOpM) and incubated for 2 hrs. Medium was removed and the monolayers were w ashed twice using PBSA before being pelleted. DNA was extracted by phenol cholorform precipitation as described, and dried overnight at 37^C before being dissolved in 500|il 0.2% nitric acid. lOOpl aliquots were used for AA analysis as describe in (Koberle et al, 1997).

Oligonucleotide Synthesis.

Oligonucleotides were synthesized by Dr Y. Z. Xu University College London, London, UK.

Duplex oligonucleotides containing G-T, O^-meGua-T, or a single unpaired C were prepared according to (Griffin et a l, 1994) and (MacPherson et al., 1999). The principle for chemical synthesis of the 34-mer oligomer containing

carboxymethylated thioguanine

(5'AGCTTGGCTGCAGGTXGACGGATCCCCGGGAATT-3', where X is S^- carboxymethylated guanine) is described in (Xu, 1998). Basically, a 34-mer

containing 6-thioguanine in position X was first prepared as described in (Xu et al.,

1992). Then, two OD (A26O) units of purified thioguanine-containing 34-mer was dissolved in 300|li1 of 0.4M phosphate buffer (pH 9.2 ) and treated with 50|il of O.IM sodium 2-iodoacetate. The carboxymethylation reaction was m onitored by FPLC by which the starting oligomer (thioguanine-oligomer) and the product oligomer (carboxymethyl-thioguanine oligomer) were well separated (Xu & Swann, 1992). The entire reaction was complete within 4 hrs. The product peak was isolated by FPLC and desalted w ith NAP-10 (Pharmacia).

^2p labelling of oligonucleotides.

Oligonucleotides were 5' ^^P end-labelled using T4 polynucleotide kinase as described by (Sambrook et al, 1989). Labelled oligonucleotides were separated from unincorporated [^^P-ATP] by Sephadex G50 spin column chromatography.

Bandshift Analysis.

Bandshift experiments were carried out by P. MacPherson in the laboratory.

Bandshift analysis using duplex ^^P-labelled oligonucleotides, formed by annealing 5' end-labelled 34-mer oligonucleotides to an excess of the appropriate complementary strand, were carried out as described in (MacPherson et al., 1999). Purified hM utSa (MacPherson et al., 1999) was incubated for 5 mins at room tem perature with 2 pmol of perfectly matched non-radioactive duplex in 20|l i1

Hepes. KOH pH8, 0.5mM EDTA, O.lmM Z nC l2,10% glycerol, 50pg poly(dl.dC). The radiolabelled duplex (20 fmol) was then added and incubation continued for a

further 20 mins. Aliquots were analyzed by electrophoresis on non-denaturing 6% polyacrylamide gels and the reaction products detected by autoradiography.

Western Blotting.

Cell extracts were prepared by incubating cell pellets with extraction buffer (1% NP40, lOmM NaF, lO pg/m l leupephn, lO pg/m l aprotinin Im M AEBSF in a 10% (v /v ) solution of PBSA) on ice for 1 hr before pelleting using a benchtop centrifuge. Supernatants were snap frozen using dry-ice and stored at -80^C until required. Western blotting for the mismatch repair proteins hMSH2, hPMS2 and hM LH l were carried using mouse monoclonal antibodies (Pharmingen USA) all at Ip g /m l. A rabbit polyclonal anti-hMSH6 antibody was kindly provided by Dr J. Jiricny (Institute for Medical Radiobiology, Zurich, Switzerland.) and used at

1/3000 dilution. Anti-PCNA (mouse monoclonal) was used at Ip g /m l (Pharmingen USA). Anti-p53 and anti-p21^^^P^/ w afl antibodies (Oncogene Research Products USA) were used at 2.5pg/m l and Ip g /m l respectively.

For western blots, 50pg of nuclear extracts were loaded per well in either 8% (for mismatch repair proteins) or 12% (for p53 and p2ic ip l/w a f l) SDS-PAGE gels along with Kaleidoscope prestained molecular weight standards (Biorad).

Separated proteins were transferred to PVDF membranes (Immobilon USA) for 45 mins-1 hr at 18V using a transblot semi-dry transfer cell (Biorad). Membranes were blocked for 1-3 hrs at room temperature with a 5% solution of pow dered milk in TBS-Tween (0.05% Tween), before being incubated with the appropriate prim ary antibody. Membranes were washed three times with TBS-Tween (0.05% Tween) prior to, and after, each antibody incubation. Immunoreactive proteins were

visualized w ith the Amersham HRP detection kit using the secondary antibodies provided.

Lymphoblastoid Growth Curves.

Lymphoblasts were seeded from exponentially growing stocks in 24 well plates at 1-2x10^ cells/w ell in 2 ml m edium and allowed grow for 24 hrs prior to treatment. A lOmM stock of N-nitroso-N-methylurea (MNU) was m ade up in 4mM potassium acetate buffer PH4. 2-amino-6-mercaptopurine (6-TG) was m ade up to a 5mM stock in O.IM potassium hydroxide, and a 20mM stock of 8-Azaguanine (8- AG) was also made up in O.IM potassium hydroxide. 5mM stocks of both

azaserine and 5-diazo-6-oxonorleucine were m ade up in PBSA. A 50mM solution of hypoxanthine was m ade in O.IM potassium hydroxide. Cisplatin (at lOmM) was dissolved in a minimum volume of dimethylsulphoxide and then m ade up to the correct volume in the appropriate culture medium. Lymphoblast cell lines were treated with different concentrations of these drugs on day 1 after seeding.

Treatment w ith cisplatin was for 1 hr before the cells were gently pelleted, washed and resuspended in fresh medium. Exponential growth in untreated control wells was m aintained by dilution.

Colony Formation Assays.

Cells were seeded from exponentially growing stock cultures at between 200-10^ cells/lOcm plate and allowed attach for 3-4 hrs prior to drug treatment. After addition of the specific drug, cells were incubated for 10-14 days and colonies counted after being fixed with methanol and stained with Giemsa. For MNU, 6- TG, 8-AG and azaserine, the drug was not removed. For cisplatin, drug treatm ent

was for 1 hr. For mitomycin C treatment was for 1 hrs and for mechlorethamine (HN2) treatment was for 30 mins. Both mitomycin C and HN2 solutions were prepared fresh each time in H2O. For UV survivals, m edium was removed and the cells were irradiated at a fluence of IJ/m ^ per second for various times using a 254nm germicidal lamp calibrated with a Latajet dosimeter.

M utation Frequency and Rate.

Mutation frequency at the HPRT locus was usually determined in 96 well plates. Cells were seeded at 10^ per well (200p,l) in ten plates (10^ cells approx) in 30|iM 6-TG. The cloning efficiency of each line was determined by plating cells at 3 /w ell in 96 well plates. Cloning efficiency was estimated using the equation

CE= -ln(fraction of -ive w ells)/(N o cells per well). M utation frequency was estimated using the equation

MF=-ln(fraction -ive wells)/(No cells per well)(CE).

Determination of the mutation rate (p) at the same locus was by fluctuation analysis. Several (between 12-20) independent clones from each cell line, established by serial dilution in 96 well plates, were grown up to a defined cell num ber and seeded in a separate 96 well plate in 30|liM 6-TG. Rate (p) was estimated using the equation

)i= MC"^ln2.

M= mutations per culture which= -InPo Po= probability of -ive cultures

Flouresence Associated Cell Sorting (FACS) Analysis.

FACS analysis was carried out with the aid of Mr Arron Rae and Mr Derek Davies of the Imperial Cancer Research Fund FACS Laboratory, Lincoln's Inn Fields London, UK.

FACS analysis of UV-irradiated or drug treated cells was determined by propidium iodide staining. Cells (10^-10^) were seeded from exponentially growing populations and allowed attach overnight. Following treatment, cells were harvested at specific time points to monitor cell cycle distribution in response to the various treatments. After harvesting cells and washing twice in PBSA, they were fixed in 1ml of 70% ethanol and stored for up to seven days at 4^C. For