3416400799

NF-kB and radioscnsi(ivity • C. DlDELOT et al. 1355

mediated transcriptional activation and provides a fcedback mechanism.

Because NF-kB was activated in numerous cancers, it might confer intrinsic radioresistance. In this study, wc investigated the effect of modulation of the NF-kB DNA-binding activity on the intrinsic radiosensitivity of head-and-neck celi Iines. We recently reported that the cellular radiosensitivity of head-and-neck carcinoma celi lines was related to baseline apoptosis (16). In the present study, we investigated whether modulation of constitutive NF-kB ac-tivity by dexamethasone (DEX) and TNFa could modify the intrinsic radiosensitivity and the basal apoptosis of head-and-neck carcinoma celi lines.

MATERIALS ANI) METHODS

Celi culture materials were purchased from Costar (Dut-scher, Brumath, France), and culture media and additives were obtained from Life Technologies (Gibco BRL, Cergy-Pontoise, France), except fetal calf serum, which was obtained from Costar (Dutscher, Brumath, France). Recombi-nant hurnan TNFa was provided by RD Systems (Abingdon, UK). Antibodies against P50 and P65 were purchased from Santa Cruz (Tebu, Le Perray-en-YveIines, France). Ali other Chemicals were purchased from Sigma (St. Quentin Fallavier, France).

Celi culture and treatment

KB head-and-neck carcinoma celi linę was kindly pro-✓ided by A. Hanauske (Munich University, Germany) as lart of the EORTC Preclinical Therapeutic Models Group 3xchange program. The KB3 subline was derived from KB ;ells, as previously described (16). Both celi lines were »rown in a 37°C, 5% C0₂ atmosphere in 75-cm² plastic issue culture flasks in RPMI 1640 medium supplemented vith 10% fetal calf serum, penicillin (100 i.u7mL), and .treptomycin (100 jitg/mL).

Modulation of NF-kB DNA-binding activity

KB and KB3 celi lines were secded at 2.10⁴ cells/cm². ^?our days later, the cells were exposed to 1 yxM DEX for !4 h or 2.5 ng/mL TNFa for 10 min. After this treatment, he cells were culturcd in drug-free medium for 14 h.

dectrophoretic mobility shift and supershift assays

Nuclear extracts from KB and KB3 cells exposed to 'NFa or DEX were prepared as described previously 17), with the following modifications. The nuclei were esuspendcd in 20 mM HEPES, pH 7.9, containing 450 iM NaCl, 0.2 mM EDTA, 0.15 mM MgCl₂, 25% (v/v) lycerol, 0.5 mM dithiothreitol, and 0.5 mM phenylmeth-Isulfonyl sulforidc, then homogeneized in Dounced Fisher Bioblock Scientiłic, Illkirch, France) and incu-ated for 30 min at 4°C after addition of 0.25% (v/v) Jonidet P-40. Nuclear extracts were eleared by centrif-gation for 30 min at 4°C at 17,000 g, and the resulting upernatants were supplemented with KC1 (0.1 M) and

Fig. 1. Constitutive DNA-binding activity of NF-kB in KB and KB3 cells. Nuclear extracts (5 pg) from (A) KB and (B) KB3 were ineubated with radiolabclcd NF-kB binding site. The DNA/NF-kB complcx was analyzed in nativc polyacrylamide gcls. This picture was representative of threc independent experiments.

immcdiately frozen in liquid nitrogen and stored at -20°C until used for electrophoretic mobility shift assay and supershift assay.

Nuclear protein concentration was determined according to Lowry et al. (18) using bovine serum albumin as standard, with two dilutions for each sample (l/10^c and l/20^e) and two determinations for each dilution. The mcan of these four concentrations was determined, and extracts were used only if the standard dcviation (SD) was less than 10%.

Distinct oligonucleotides containing complemcntary se-quences at their 3' termini and binding motifs for NF-kB were annealed and the protruding single-stranded regions filled in with (a-³²P) dCTP (Amersham Pharmacia Biotech, Orsay, France) using Klenow fragment of DNA polymerase I (Promega, Charbonnieres, France). The sequences of the oligonucleotides used were as follows: 5'-AGTTGAGGCL GAC3TT-3'; 3'-CęCTGAAAGGGTCCG-5^/.

The binding reaction mixture (10 pL) for gel retardation assays contained 1 ng of DNA probc, 2 pg poly'dI-dC, and 5 pg of the different nuclear protein extracts. The mixturc was ineubated for 30 min at 4°C. The DNA protein com-plexes were resoWed in native 5% polyacrylamide gels in 0.5 X Tris-borate ethylcnediaminetetraacetic acid (EDTA) at 100 V for 45 min. The dried gels were exposcd to Trimax