[Identification of nucleolar localization signal sequence of tumor metastasis suppressor gene-1].

Objective: To identify the putative specific localization signal sequence of tumor metastasis suppressor gene-1 (TMSG-1) and to explore the mechanism of subcellular localization of TMSG-1 protein.

Methods: Vectors expressing green fluorescence protein (GFP) tagged different TMSG-1 fragments were generated and transfected into human embryo kidney 293 (HEK293) cells. The expression of those fusion proteins was detected by Western blotting and their subcellular localizations were observed by laser confocal microscope.

[Transcriptional activation of TMSG-1 by complex of KLF6 and Sp1].

Objective: To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1.

Methods: Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1.

[Influence of phosphoprotein associated with glycosphingolipid microdomains 1 on biologic behavior of human prostatic cancer cell line in-vitro].

Objective: To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8.

Methods: The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein.

[Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro].

Objective: To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro.

Methods: Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation.

[Overexpression of human tumor metastasis-related gene TMSG-1 suppresses cell proliferation and invasion of a highly metastatic prostate cancer cell line PC-3M-1E8 in vitro].

Objective: To investigate the effects of tumor metastasis-related gene TMSG-1 overexpression on the proliferation and invasion of a highly metastatic prostate cancer cell line in vitro.

Methods: The eukaryotic expression plasmids containing full-length TMSG-1 cDNAs were stably transfected into the highly metastatic prostate cancer cell line PC-3M-1E8. Clones highly expressing TMSG-1 were identified by RT-PCR and Western Blot analysis after G418 screening.

[Correlation of expression of RhoC with invasiveness of breast cancer cells in vitro].

Objective: To investigate the expression of RhoC in breast cancer cells with different metastatic potential and its correlation with invasiveness.

Methods: Expression of RhoC mRNA and protein in human breast cancer cells MCF-7 with low metastatic potential and MDA-MB-231 with high metastatic potential was detected by RT-PCR, Western blot, immunohistochemistry and immunofluorescence staining. Eukaryotic expression plasmids of RhoC were constructed and transfected into MCF-7 cells.

[Correlation of expression of RhoC with invasiveness of prostate cancer cell line PC-3M in vitro].

Objective: To investigate the expression of RhoC in prostate cancer cells with different metastatic potential and the correlation with invasiveness.

Methods: Expression of RhoC mRNA and protein in human prostate cancer cell subclones PC-3M-2B4 with non-metastatic potential and PC-3M-1E8 with highly metastatic potential was detected by RT-PCR and Western blot. Eukaryotic expression plasmids of RhoC were constructed and transfected into PC-3M-2B4.

[Overexpression of tumor metastasis suppressor gene 1 suppresses proliferation and invasion, but enhances apoptosis of human breast cancer cells MDA-MB-231 cells].

Objective: To investigate the effects of tumor metastasis suppressor gene 1 (TMSG-1) overexpression on the proliferation, invasion and apoptosis of breast cancer cells and to determine possible correlations of TMSG-1 and metastasis of breast cancer.

Methods: Full-length human TMSG-1 coding sequences were cloned into plasmid pcDNA3.0-FLAG.

[Biological implications of the inhibition of survivin by RNA interference in human androgen-independent prostate carcinoma with highly metastatic potential].

Objective: To determine the expression level of survivin in androgen-independent prostate carcinoma, and to investigate the biological role of survivin in invasion and metastasis of androgen-independent prostate carcinoma.

Methods: Highly metastatic prostatic cancer cell line PC-3M-1E8 was stably transfected with pSilencer plasmid targeting survivin expression by RNA interference. The biological effects were observed, including anchorage-independent growth, in vitro invasion by soft agar colony formation and Boyden chamber assay, and also in vivo tumorigenesis in nude mice.

[Monoclonal antibody against G3BP: preparation, characterization and its application in analysis of human tumors].

Objective: To better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.

Methods: By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG.

[Monoclonal antibodies against human tumor metastasis suppressor gene-1 TMSG-1: preparation, characterization and application].

Objective: In order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors.

Methods: A dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice.