Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation

• Published in: The Journal of biological chemistry Vol. 263; no. 1; pp. 254 - 261
• Main Authors: Troen, B R, Ascherman, D, Atlas, D, Gottesman, M M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05.01.1988; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cathepsin L; Cathepsins - genetics; Cell differentiation, maturation, development, hematopoiesis; Cell Line; Cell physiology; Cells, Cultured; Cloning, Molecular; Cosmids; Cysteine Endopeptidases; DNA Restriction Enzymes; Endopeptidases; Enzyme Precursors - genetics; Fibroblasts - enzymology; Fundamental and applied biological sciences. Psychology; Genes; Genetic engineering; Genetic technics; Humans; Lysosomes - enzymology; Methods. Procedures. Technologies; Mice; Molecular and cellular biology; Molecular cloning; Nucleic Acid Hybridization; Transfection; Human; Cell culture; Cathepsin L; Rodentia; Gene library; Gene expression; Precursor; Screening; Vertebrata; Mammalia; Transfection; Major excreted protein; Mouse; Radioimmunoassay; Restriction map; Tumor; Molecular cloning; Southern blotting; Fibroblast
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)57386-0

The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.