Monoclonal antibodies against human tumor metastasis suppressor gene-1 (TMSG-1): preparation, characterization, and application.
To better understand the molecular mechanism of metastasis, the monoclonal antibody against tumor metastasis suppressor gene-1 (TMSG-1, one novel gene) was prepared, characterized, and applied in estimating the metastatic potential of human tumors. A dominant epitope, TMSG-1(15)--derived from TMSG-1--was automatically synthesized based on the Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen solution was used to immunize BALB/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and their characterization was performed by Western blotting and immunohistochemical staining. The hybridoma cell line secreting anti-TMSG-1 antibody, designated C8, was established after primary ELISA screening and consequent rapid limited dilution. C8 was IgM in isotyping. The competitive inhibition assay showed that the antibody was TMSG-1 specific. Furthermore, in Western blotting, a protein band of about 45 kD was detected with nonmetastatic variant PC-3M-2B4 and PG-LH7 cells, but not with the isogenetic metastatic variant PC-3M-1E8 and PG-BE1 cells. Immunohistochemistry method showed that positive staining presented in the cytomembrane and cytoplasm of 2B4 and LH7 cells, while 1E8 and BE1 cells were non-reactive. The sections from paraffin-embedded blocks of human cancer (52 cases of breast carcinoma and 41 cases of colon cancer) were stained, showing strongly positive in non-metastatic tumor and weakly positive or negative in metastatic tumor; the strongly positive rate of TMSG-1 expression in human cancers were, respectively, 36%, 7.4%, 52.4%, and 35% in non-metastatic and metastatic breast cancer and non-metastatic and metastatic colon cancer (p = 0.02). The monoclonal antibody developed against synthetic peptide was TMSG-1 specific, and it has promise in Western blot and immunohistochemistry for detecting the TMSG-1 expression of cancer cells and tissues. It may provide an important tool in the study of TMSG-1 expression and function in both experimental and clinical studies. Furthermore, our tests confirmed that TMSG-1 protein has excellent inverse correlation to tumor metastasis potential, with the molecular weight of 45 kD (supporting the encoded protein containing 380 amino acids) and localization in cytomembrane and cytoplasm of tumor cell.
Fei
Pei
P
Department of Pathology, Health Science Center, Peking University, Beijing, China.
Junyu
Ning
N
Jiangfeng
You
Y
Jingpin
Yang
Y
Yuping
Wang
W
Zhihui
Han
H
Jieliang
Wang
W
Xianglin
Cui
C
Shaomin
Yang
Y
Jie
Zheng
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D051049:Sphingosine N-Acyltransferase; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D025521:Tumor Suppressor Proteins; C438311:CERS2 protein, human; D051049:Sphingosine N-Acyltransferase