Gene silencing by CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. It has been shown previously that Betaig-h3 gene, which encodes an extracellular matrix protein involved in cell adhesion and tumorigenesis, is down-regulated or silenced in a variety of human cancer cell lines. To unravel the underlying molecular mechanism(s) for this phenomenon, DNA methylation patterns of Betaig-h3 CpG island were examined in normal, immortalized, and cancer cell lines derived from lung, prostate, mammary, and kidney. A good correlation was observed between promoter hypermethylation and lost expression of Betaig-h3 gene, which was supported by the data that demethylation of promoter by 5-aza-2V -deoxycytidine reactivated Betaig-h3 and restored its expression in Betaig-h3-silenced tumor cell lines. This result was further substantiated by a luciferase reporter assay, showing the restoration of promoter activities and increased response to transforming growth factor-B treatment in Betaig-h3-negative 293T cells when transfected with unmethylated Betaig-h3 promoter. In contrast, activity of Betaig-h3 promoter was completely inactivated by in vitro methylation. Furthermore, CpG methylation of Betaig-h3 promoter was also shown in primary lung tumors that expressed decreased level of Betaig-h3 protein. These results suggest that promoter methylation plays a critical role in promoter silencing of the Betaig-h3 gene in human tumor cells.

High-energy (HZE) heavy ions, when compared to low-LET radiation, are highly effective in inducing gene mutation, chromosomal aberrations and neoplastic transformation. However, the underlying molecular mechanisms are not clearly understood. We have recently shown that the down-regulation of Betaig-h3 expression is causally linked to the tumorigenic phenotype of papillomavirus-immortalized human bronchial epithelial (BEP2D) cells treated with high-LET alpha-particle radiation. Using the BEP2D cell culture system, a radiation-induced transformation model has been established by a single 60-cGy dose of (56)Fe heavy-ion radiation. To determine whether the Betaig-h3 gene is involved in (56)Fe ion-induced tumorigenesis, the expression levels of the Betaig-h3 gene in tumorigenic cell lines and the ability of in vivo tumor suppression through the reintroduction of the Betaig-h3 gene in tumorigenic cells were determined. We found that the expression level of this gene is markedly decreased in three tumorigenic cell lines ((56)FeT1-T3) compared with parental BEP2D cells. Ectopic expression of its cDNA in the (56)FeT2 tumorigenic cells significantly suppressed their tumorigenicity. Although biologically active TGFB1 is elevated in two of three tumorigenic cell lines, all these cell lines are resistant to the induction of Betaig-h3 expression by incubating the transformed cells with exogenous TGFB1 relative to control cells. Our data strongly suggest that down-regulation of Betaig-h3 expression results from the defect in the TGFB1 signaling pathway and plays a pivotal role in the tumorigenic process induced by (56)Fe heavy-ion radiation.

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