Immunological quantitation of levels of tissue inhibitor of metalloproteinase-1 in human colon cancer.
Treatment of rodent cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate activates protein kinase C, leading to increased expression of several genes, including a gene originally designated TPA-S1 or phorbin (M. D. Johnson, G. M. Housey, P. T. Kirschmeier, and I. B. Weinstein, Mol. Cell Biol., 7: 2821-2829, 1987). Sequence analysis of this cloned gene indicated homology with human erythroid-potentiating activity and tissue inhibitor of metalloproteinase (TIMP-1). Elevated levels of phorbin mRNA have been observed in human colon tumors (J. G. Guillem, M. F. Levey, L. L. Hsieh, M. D. Johnson, P. LoGerfo, K. A. Forde, and I. B. Weinstein, Mol. Carcinogen., 3: 68-74, 1990) and this increase correlated with the extent of invasion. To further investigate this phenomenon at the protein level, monoclonal antibodies were developed against the recombinant form of TIMP-1. A competitive enzyme-linked immunosorbent assay was developed for quantitation of the TIMP-1 protein in tissue extracts. Elevated levels of TIMP-1 protein were found in 31 human colon tumors, compared to paired samples of adjacent normal mucosa. In a subset of samples, previously analyzed for phorbin mRNA levels (n = 25), there was a good correlation between the abundance of TIMP-1 protein and phorbin mRNA. Immunoaffinity column purification of tumor extracts followed by Western blot analysis was used to confirm the enzyme-linked immunosorbent assay data. These results provide evidence that phorbin and TIMP-1 represent the same gene. In addition, the immunoassays we have developed may be useful in further studies on the role of TIMP-1 in human colon cancer.
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Comprehensive Cancer Center, Columbia University, New York, New York 10032.
Levy
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Weinstein
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Santella
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RM
D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D019714:Tissue Inhibitor of Metalloproteinases
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D019714:Tissue Inhibitor of Metalloproteinases