To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylation sites, 2 casein kinase II phosphorylation sites and 1 N-myristoylation site. The pattern of TMSG-1 expression in 6 types of human tumor tissues indicated levels of transcripts were the highest in prostate carcinoma. TMSG-1 had lower expression in metastases of lung carcinoma compared to primary lung carcinoma. Similarly the expression levels were higher in well-differentiated colon carcinoma than that in poorly differentiated colon carcinoma. TMSG-1 could also be detected in breast, ovarian, and pancreatic carcinoma. In 9 samples of primary gastric carcinoma tissues, RT-PCR and densitometric analysis demonstrated TMSG-1 expression levels in samples with lymph node metastases had a decreased tendency, compared to those without lymph node metastases. The difference was significant by student's t test (p<0.05). These results indicated TMSG-1 expression levels were inversely correlated with tumor metastatic potential.
TMSG-1 was a tumor metastasis-related gene identified using mRNA differential dis-play, whose expression level was lower in cancer cell lines with higher metastatic potential and in tumor tissue with metastasis. TMSG-1 was transfected to prostate cancer cell line (PC-3M-1E8) with high metastatic potential to observe the effects of increased expression of TMSG-1 on V-ATPase activity, intracellular pH and cell apoptosis. Subcellular localization of the encoded pro-tein of TMSG-1 was determined by using GFP. Results showed that there were no differences of V-ATPase activity among parental PC-3M-1E8 cell line, pcDNA3 transfectant and anti-TMSG-1 transfectant, whereas the V-ATPase activity was significantly higher in TMSG-1 transfectant than that in parental PC-3M-1E8 cell line, pcDNA3 transfectant and Anti-TMSG-1 transfectant (p<0.001). Intracellular pH (pHi) was detected by using the pH-dependent fluorescence probe BECEF. Re-sults showed the pHi was significantly increased in TMSG-1 transfectant. Cell apoptosis assay demonstrated cell apoptosis was significantly higher in -1 transfectant (p<0.01) and BCL2 expres-sion was down regulated. Subcellular localization of TMSG-1 protein showed TMSG-1 was a transmembrane protein, which predicted TMSG-1 protein was located in cytoplasm system, such as endoplasmic reticulum and mitochondrial. These results indicated TMSG-1 up regulation in prostate cancer cell line could promote V-ATPase activity, increase pHi and cell apoptosis, and inhibit the expression of BCL2.
