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. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining.
Results: One hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05).
Conclusion: C8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.
Download full-text PDF |
Source |
|---|
Publication Analysis
Top Keywords
Similar Publications
[Expression and significance of Ki-67, PI3K and Beclin1 in oral squamous cell carcinoma].
Shanghai Kou Qiang Yi Xue
February 2020
Department of Stomatology, Nanjing Tongren Hospital, School of Medicine, Southeast University. Nanjing 211102, China.
Purpose: To detect the expression and significance of Ki-67, PI3K and Beclin1 in oral squamous cell carcinoma (OSCC).
Methods: Thirty patients with OSCC admitted to Nanjing Stomatology Hospital from January 2017 to December 2018 were selected. All specimens were harvested and treated with immunohistochemical staining.
Silencing of LASS2/TMSG1 enhances invasion and metastasis capacity of prostate cancer cell.
J Cell Biochem
April 2014
Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, P.R. China; Department of Pathology, School of Basic Medical Sciences, Inner Monglia Medical College, Huhhot, 010059, P.R. China; Department of Pathology, The Affiliated Hospital of Inner Monglia Medical College, Huhhot, 010059, P.R. China.
Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), was firstly cloned by our laboratory in 1999. However, its antitumor molecular mechanisms are still unclear. LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar H(+) ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase.
View Article and Find Full Text PDFSilencing of a novel tumor metastasis suppressor gene LASS2/TMSG1 promotes invasion of prostate cancer cell in vitro through increase of vacuolar ATPase activity.
J Cell Biochem
July 2012
Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, PR China.
Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), is a newly found tumor metastasis suppressor gene in 1999. Preliminary studies showed that it not only suppressed tumor growth but also closely related to tumor metastasis, however, its molecular mechanisms is still unclear. There have been reported that protein encoded by LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase.
View Article and Find Full Text PDF[Identification of nucleolar localization signal sequence of tumor metastasis suppressor gene-1].
Zhonghua Bing Li Xue Za Zhi
November 2011
Department of Pathology, Health Science Center, Peking University, Beijing 100191, China.
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].
Zhonghua Bing Li Xue Za Zhi
August 2011
Department of Pathology, Peking University, Beijing, China.
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.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!