[Show abstract][Hide abstract] ABSTRACT: TMSG-1 was a tumor metastasis-related gene identified using mRNA differential display, 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 protein 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. Results 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 expression 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.
No preview · Article · Jan 2004 · Science in China Series C Life Sciences
[Show abstract][Hide abstract] ABSTRACT: To observe the relationship between TMSG-1 gene and tumor metastatic phenotype.
TMSG-1 cDNA fragment which contained full length open reading frame of TMSG-1 gene was cloned into pcDNA3 plasmid to reconstruct sense and antisense eukaryotic expression plasmids of TMSG-1 gene containing neo selection marker. Both sense and antisense eukaryotic expression plasmids of TMSG-1 gene were transfected into the highly metastatic subclone PG-BE1 by LipofectAMINE method and the positive clones were selected by G418. RT-PCR was used to examine the expression level of the transfected gene and the changes of biological characteristics were checked by a series of in vitro and in vivo assays.
The results showed that higher expression levels of TMSG-1 gene in BE1-S cells (cells transfected by sense TMSG-1 cDNA) than the control BE1 cells and BE1-V cells (cells transfected by pcDNA3 plasmid). The expression levels of TMSG-1 gene in BE1-AS cells (cells transfected by antisense TMSG-1 cDNA) were lower than those of the control BE1 cells and BE1-V cells. Compared with the control BE1 cells and BE1-V cells, BE1-AS cells grew more rapidly, and produced more foci in soft agar. Although the BE1-S cells did not reveal significant different growth capacity, the infiltrating ability and colony formation potential of BE1-S cells were decreased, compared with the control BE1 cells and BE1-V cells. Flow cytometry showed higher percentage of BE1-S cells in G0G1 phase than that of BE1 cells, and the presence of apoptotic peak in BE1-S cells.
The results suggest TMSG-1 gene may represent a tumor metastasis suppressor gene.
No preview · Article · Mar 2003 · Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences
[Show abstract][Hide abstract] ABSTRACT: 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 fulllength 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 nonmetastatic cell line 2B4. The difference was significant. Fulllength 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 Nmyristoylation site. The pattern of TMSG1 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 welldifferentiated 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.
No preview · Article · Nov 2002 · Science in China Series C Life Sciences