SUMMARY: Nuclear factor-B (NF-B) is a transcriptional regulator of inducible expression of genes includingcyclooxygenase-2 (COX-2), regulating cell proliferation. NF-B is kept silent in the cytoplasm via interaction with the inhibitory protein IB␣ and transmigrated into the nucleus upon activation. However, constitutive NF-B has been found in the nucleus of some cancer cells. We investigated the role of NF-B in COX-2 expression and cell proliferation in human gastric cancer AGS cells. AGS cells were treated with antisense oligodeoxynucleotide (AS ODN) or sense oligodeoxynucleotide (S ODN) for the NF-B subunit p50, or they were transfected with a mutated IB␣ gene (MAD-3 mutant) or a control vector, pcDNA-3. AGS cells were treated with COX-2 inhibitors such as indomethacine and NS-398 or prostaglandin E 2 . mRNA expression for COX-2, and protein levels for p50, IB␣, and COX-2 were determined by reverse transcription polymerase chain reaction and Western blot analysis. The NF-B levels were examined by electrophoretic mobility shift assay. Thromboxane B 2 (TXB 2 ) and 6-ketoprostaglandin F 1␣ (6-keto-PGF 1␣ ) levels were determined by enzyme-linked immunosorbent assay. Cell proliferation was assessed by viable cell counting, [ Cell proliferation, mRNA expression and protein level of COX-2, and production of TXB 2 and 6-keto-PGF 1␣ were inhibited in cells treated with AS ODN or transfected with the mutated IB␣ gene, which had lower NF-B levels than cells treated with S ODN or transfected with control vector. COX-2 inhibitors suppressed cell proliferation and production of TXB 2 and 6-keto-PGF 1␣ , in a dose-dependant manner. Prostaglandin E 2 prevented the inhibition of proliferation in cells treated with AS ODN or transfected with the mutated IB␣ gene. In conclusion, NF-B mediates COX-2 expression, which may be related to cell proliferation, in human gastric cancer cells. (Lab Invest 2001, 81:349 -360).