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Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface
John D Hooper1, Andries Zijlstra, Ronald T Aimes
1Department of Cell Biology, The Scripps Research Intitute, La Jolla, CA 92037, USA.
Abstract:
We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M(+)HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.
Insights
Researchers identified SIMA135/CDCP1, a cell surface protein, using subtractive immunization. Its expression correlates with cancer metastasis and it is tyrosine phosphorylated, suggesting a role in tumor progression.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Subtractive immunization (SI) is a method to generate antibodies against specific cell targets.
- The highly metastatic human epidermoid carcinoma cell line M(+)HEp3 expresses unique proteins.
- Characterizing these proteins can lead to new cancer biomarkers.
Purpose of the Study:
- To identify and characterize a novel cell surface protein, SIMA135/CDCP1, expressed by M(+)HEp3 cells.
- To investigate the correlation between SIMA135/CDCP1 expression and cancer cell metastatic potential.
- To explore the post-translational modifications and expression patterns of SIMA135/CDCP1.
Main Methods:
- Monoclonal antibody (mAb) generation using subtractive immunization.
- Immunopurification and proteomic analysis for protein identification.
- Immunocytochemistry, Western blotting, and Northern blotting for expression and characterization.
- Analysis of protein glycosylation and phosphorylation.
Main Results:
- SIMA135/CDCP1 was identified as a 135 kDa cell surface protein.
- Its expression levels correlated with the metastatic ability of HEp3 cell variants.
- SIMA135/CDCP1 is N-glycosylated, tyrosine phosphorylated (Src kinase involvement), and detected in various tumor cell lines.
- mRNA expression is highest in skeletal muscle and colon; protein is found on normal colon epithelial cells and dysregulated in colon tumors, with evidence of shedding.
Conclusions:
- SIMA135/CDCP1 is a novel, potentially metastatic cell surface antigen.
- Its phosphorylation and expression patterns suggest a role in cancer progression.
- SIMA135/CDCP1 may serve as a diagnostic or therapeutic target.