Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.

AIM: To identify esophageal cancer related gene2 (ECRG2) associated proteins and their possible interactions with ECRG2 gene. METHODS: In the yeast forward two-hybrid system, ECRG2 was fused with the DNA-binding domain (DBD) of Gal4 and human fetal liver cDNA library was fused with the transcriptional activation domain (AD) of Gal4. We performed a high-stringency scale procedure to screen ECRG2 against human fetal liver cDNA library and characterized positives by sequence analysis. RESULTS: We found the following 9 putatively associated proteins. They were metallothionein2A, metallothionein1H, metallothionein1G, ferritin, erythrocyte membrane protein band4.2, mitochondrial ribosomal protein S12, hypothetical protein FLJ10101, and a novel gene whose cDNA was found to have no strong homology to any other previously characterized gene whose DDBJ/EMBL/GenBank accession number is AF422192 mapped to human chromosome 14q31. CONCLUSION: MT, a potential interaction partner for ECRG2, might be involved in the regulation of cell proliferation and apoptosis, and in various physiological processes. Determination of a reliability score for each single protein-protein interaction, especially interaction of ECRG2 and MT, permits the assignment of ECRG2 and unannotated proteins to biological pathways. A further understanding of the association between ECRG2 and MT should facilitate the functions of ECRG2 gene.