The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3.
The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Alper
O
O
Growth Factor Division, National Cancer Center Research Institute, Tokyo, Japan.
Yamaguchi
K
K
Hitomi
J
J
Honda
S
S
Matsushima
T
T
Abe
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001483:Base Sequence; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D018719:Erb-b2 Receptor Tyrosine Kinases; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003470:Culture Media; D003545:Cysteine; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D013462:Sulfur Radioisotopes