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1356

I. J. Radiation Oncology • Biology • Physłcs

Volumc 51, Numbcr 5, 2001

Fig. 2. Analysis of components in the NF-kB complex by supershift cxperimcnts. Nuclearextracts (5 /xg) preparcd from (A) KB or (B) KB3 untreatcd cells werc ineubated willi anlibodics raiscd against (2) P50 or (3) P65 or (1) without aniibody, bcforc the addilion of Ihe radiolabelcd NF-kB binding sile. Reaction mixiure was rcsoWcd in nativc polyacrylamide gcls. Tlicse pictures wcrc reprcscntativc of ihree independent cxperimcnts.

XM (Kodak) film overnight at —80°C. To test the specifie-ity of the binding, competition studies were performed in the presence of a 100-fold molar excess of the spccific unlabeled probe beforc the addilion of 1 ng of the radiola-beled probe. Supershift assays wcrc carried out with 1 pg of antibodies raiscd against cither P50 or P65 (Santa Cruz, Tebu, France) ineubated for 15 min at room temperaturę with nuclcar proteins beforc the addition of the labeled probe.

Detection of apoptosis

Cells were sceded at 2.10⁴ cells/mL in 75-cm² plastie tissue culture flasks. Four days latcr, cells were ineubated in 5 mM EDTA for 5 min and washed with phosphatc-buffered salinę. 10⁵ cells were ineubated with 100 pL of IX binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCI₂), 5 pL annexin V-FITC (PharMingen, Becton Diekinson), and 2 pL propidium iodide (50 pg/mL). After 15 min at 4°C in the dark, cells were suspended in 400 pL of binding buffer and analyzed by flow cytometry (FACSealibur, Becton Diekinson) us-ing Celi Qucst software (Becton Diekinson).

Radiosensitivity

The radiosensitivity of each celi linę was determined by using soft agar elonogenic assays aceording to the method described by Griffon Etienne et al. (19). Briefly, cells were trypsinized, harvestcd, and sceded in 0.3% soft agar (Bacto Agar, Difeo, Detroit, MI) at 2.10³ and 3.10³ cells/well for the KB and KB3 celi linę, respectivcly. After 1 h ineubation, cells were irradiated (0.5-10 Gy) using Theratron 780c ⁶⁰Co unit (Theratronics, Ottawa, Canada). After 14 days of ineubation, the colonies were semi-automatically counted using image analysis (Inspector, Matrox, Canada). Only colonies with a mean diameter above 50 pm were counted. The results were normalized to unirradiated Controls. The cxperi-mental dosc-response eurvcs were fitted using the linear-quadratie model, (S = t~^al> D ) and the radiosensitivity parameters «, /3, and SF₂ (surviving fraction at 2 Gy) were calculated aceording to Thames et al. (20). Data shown resulted from a minimum of thrce independent experiinents.

Statistical analysis

Data are mean values ± (SD), calculatcd from at least thrce independent expcriments. Comparisons were madę using the Mann-Whitney U test. Statistical significancc was p < 0.05.

RESULTS

Constitutive NF-kB DNA-binding actmty and modulation by dexamethasone or TNFa

Nuelear NF-kB DNA-binding activity was investigated in untreated KB and KB3 cells. Figurę 1 shows that NF-kB