Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor, and loss of heterozygosity at ATBF1 locus occurs frequently in gastric cancers. We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer and that ATBF1 enhanced the promoter activity of P21(Waf1/Cip1). We also found that ATBF1 moves between cytoplasm and nucleus, but the precise mechanism of translocation is unknown. In this study, we investigated the mechanism of ATBF1 translocation to the nucleus with the runt domain transcription factor 3 (RUNX3) in cooperation with TGF-beta signal transduction. Materials and methods: To analyze the expression of ATBF1 and RUNX3 in gastric cancer cells, we performed immunohistochemistry on 98 resected gastric cancer tissue samples and scored the nuclear staining intensity as grade 0 to grade 5. Co-immunoprecipitation (co-IP) of ATBF1 and RUNX3 was. performed. Dual luciferase assays were performed by transfecting ATBF1 and RUNX3 with a p21(Waf1/Cip1) reporter vector. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3 in response to TGF-beta signal, we examined the subcellular localization of ATBF1 and RUNX3 in gastric cancer cells treated with recombinant TGF-beta 1 using confocal laser scanning microscopy. Results: Strong immunohistochemical nuclear staining of ATBF1 was observed in 37 (37.8%) of the gastric cancer tissue samples, and RUNX3 nuclear staining was observed in 15 (15.3%). There was a statistically significant correlation between ATBF1 and RUNX3 nuclear localization (rs = 0.433, p < 0.001). Co-IP revealed a physical association between ATBF1 and RUNX3. ATBF1 and RUNX3 up-regulated p21(Waf1/Cip1) promoter activity synergistically. In SNU16 gastric cancer cells, ATBF1 and RUNX3 were cytoplasmic before TGF-beta 1 stimulation, but after 24 h of TGF-beta 1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus in response to TGF-beta 1 signal transduction and might function in the nucleus as tumor suppressor and transcriptional regulator. (C) 2010 Elsevier Inc. All rights reserved.