3416400795

In vitro 5-fluorouracil sensitivity and Bcl-2/Bax ratio 1381

contałning mutant-type p53. In the present siudy, we investigated ihc difference in 5-FU sensitivity observed in six hunian cancer celi lines with different p53 status and tried to evidence the basis of this difference by following BcI-2/Bax ratio and its correlation with p53 status and mRNA or protein expression induction aftcr exposurc to 5-FU equitoxic concentration. Whcthcr the 5-FU sensitivity differences arc a result of different p53 functionality causing differences in Bcl-2 and Bax regulation, the implication of p53 protein cxprcssion induction in G1 arrest ability will be discussed and reconsidered.

MATERIALS AND METHODS

Materials and Chemicals

Celi culture materials wcrc purchased front Costar (Dutscher, Bruntath, France), culture media and additives front Life Technologies (Gibco BRL, Ccrgy-Pontoise, France), cxcept for fetal calf serum, which was obtained front Costar. Taq-polymcrasc, RNAsc H, random printcrs, SuperScript II® DNA polymcrase, deoxynucleotidc triphosphate were purchased front Life Technologies. Anti-bromodeoxyuridin monoclonal anti-bodics, p53 monoclonal antibodics (DO-7) and pcroxidase-conjugatcd antibodics were provided by Dako (Trappes, France). Bax (N-20) polyclonal antibodics wcrc purchased front Tebu (Le Perray-en-Yvelines, France). Ali otlier Chemicals were purchased front Sigma (St Quentin Fallavier, France) and were of molecular biology gradc.

Celi culture

CAL51 hunian breast adenocarciiloma, PANC3 panereas carci-nonta, CAL27 and CAL33 hunian head and neck carcinoma celi lines were kindly provided by Dr JL Fischel (Centrę Antoine Lacassagne, Nice, France). FaDu and KB, head and neck carcinoma celi lines, w'cre obtained from Professor A Hanauske (Munich University, Germany) as part of the EORTC Preclinical Thcrapeutic Models Group exchange program. Ali celi lines were grown in 75 cni² plastic tissue culture flasks in RPM1 1640 medium supplemented willi 10% licat inactivaled fetal calf serum, penicillin (100 iu inl '), streptomycin (100 pg nil ') in a 37°C, 5% C0₂ atmosphere. The cells were exposed at day 4 aftcr seeding to equitoxic 5-FU concentrations (IC_M,) for 24 h, tlien analysed immediately.

Cytotoxicity assay

M IT assays were carried out according to a procedurę previously reported (Barberi-llcyob et al, 1993). Briefly, cells were seeded at the initial density of 2.10⁴ cells ml"¹ in 96 well nticro titration platcs. 72 h aftcr plating, cells wcrc exposcd for 72 h to 5-FU concentrations ranging front 0.08--4.I0^J pM, each concentration being lested in scxtuplicate. 50 pl of 0.5% MTT solution were tlien added in each well and ineubated for 3 li at 37°C to allow MTT metabolization. The formazan crystals wcrc dissolved by adding 50 pl per well of 25% sodiunt dodecylsulfate solution and vigorous pipetting. Absorbance was measured at 540 mit using a Multiskan MCC/340 piąte rcader (Labsystem, Cergy-Pontoise. France). Results were expressed as relative absorbance to un-treated Controls. 5-FU concentrations yielding 50% growth inhibition (IC_VJ) were calculated using medium effect algorithm (Chou and Talalay, 1987) and exprcssed as mean values of five independent experintents.

Analysis of p53 mutations

To identify p53 genomie ntutation, direct DNA aulomated fluores-cent sequcncing analyses were conductcd. Both DNA strands wcrc sequenced. Briefly, PCR was perfornied using four pairs of primers covering cxons 2-9 and including Banking intronic splicing sites (one pair for exons 2-4, one for cxons 5 and 6, one for exon 7 and finally one pair for exons 8 and 9), in a 20 pl volume containing lOmntol l¹ Tris-HCl, 50 mmol l¹ KC1, 1.5 mmol I^-1 MgCl„ 0.2 mmol l^-1 deoxynuclcotide triphospltates, 0.5 pmol I"¹ of each primer, and I pg of genomie DNA. The reac-tions were carried out using a Perkin Elmer/Cctus thcrntal cyclcr model 9600. The PCR products wcrc then purified using Sephacryl S400IIR (Antersham-Phamtacia Biotech, Lcs Ulis, France). 5 pl of purified fragments were uscd for scquencing with a Thermo Sequena.se™ Dye Terminator Cycle Sequencing kit (Amersham-Pharmacia Biotech), using the same PCR primers. Aftcr purification with Biogel PIO (Bio Rad), the products wcrc sequenced using ABI 373 aulomated DNA sequencing system (Applied Biosystcm).

RNA isolation and RT-PCR analysis

Isolation of total RNA was perfornied using TRIzol® according to the manufacturer’s specifications (Life Technologies). cDNA syntltesis was perfornied with 1 pg total RNA in a reaction volumc of 20 pl containing 100 ng of random primers, 50 mM Tris-HCl, pH 8.3, 75 mM KCI, 3 mM MgCl,, 0.5 mM deoxynuclcotide triphosphate, 10 mM dithiothreitol and 200 units SuperScript 11® rcverse transcriptase and ineubated for 10 min at room temperaturę, 50 min at 42°C, followed by 15 ntin at 70°C. RNAsc-II (2.5 units) was added into each sample, then ineubated for 20 min at 37°C. cDNA samplcs wcrc stored at -20°C umil analysed.

p53 and p21

p53 and p2I senti-quantilative PCR analyses were then perfornied using p2-nticroglobulin ((3₂m) as reference gene. 0.5 pl or I pl of cDNA samples were ittixed, rcspcctively for p53 or p2l amplifica-tion, in a volumc of 20 pl containing I6 mM (NH₄)₂SO_t, 67 mM Tris-HCl, pil 8.8, 0.01% Tween 20, 2 or 1.5 mM MgCl, rcspec-tivcly for p53 or p21 amplification, 0.2 mM dNTP, 5 pM of each 5'- and 3^/-primers, and 0.5 unit of Taq polynterasc. I he primers sequences wcrc 5LTCTGTGACTTGCACGTACTC-3' (sense) and 5^/-CACGGATCTGAAGGGTGAAA-3^/ (antisense) for p53 (Aguilar Santeliscs et al, 1996), 5'-CCCAGTGGACAGC-GAGCAGC-3' (sense) and 5' ACTGCAGGCTTCCTGTGGGC-3' (antisense) for p2l, 5^/-ACCCCCACTGAAAAAGATGA-3^/ (sense) and 5'-ATCTTCAAACCTCCATGATG-3' (antisense) for p,-microglobulin (p₂m) (Gussow et al, 1987).

The PCR tubes were ineubated for p53 and Pm amplification, as follows: the First cycle was 5 min at 95°C, I min at 57°C and I min at 72°C. The 33 or 36 following eycles, respectively for p53 and |)21 amplification, were I min at 94°C, I min at 57°C and 1 min at 72°C. In each casc. after completion of PCR cycles, the mixture was finally ineubated for 7 min at 72°C. p53 and p, PCR products wcrc electrophoretized on 1% agarose gel containing 0.1 pg ml ¹ of ethidium bromide. Quantification was perfornied by UV transillumination using a Gel Doc 1000 system (Bio Rad,

© 2000 Cancer Research Campaign

British Journal of Cancer (2000) 83(10), 1380-1386