erbB family receptor expression and growth regulation in a newly isolated human breast cancer cell line.
A new human breast cancer cell line (SUM-52PE), originating from a malignant pleural effusion specimen, that can be cultured under serum-free conditions has been isolated. Experiments were conducted to examine the relationship between expression of the erbB family of growth factor receptors and growth regulation in these cells. SUM-52PE cells are epidermal growth factor receptor negative but express single copy levels of erbB-2 protein. Southern blot analysis indicates that the erbB-2 gene is not amplified in these cells. The cells also express mRNA for both erbB-3 and erbB-4. Phosphotyrosine Western blot analysis of membrane protein obtained from SUM-52PE cells indicates the presence of a constitutively tyrosine phosphorylated M(r) 185,000 protein. Immunoprecipitation, using antibodies to erbB-2 or erbB-3, coupled to phosphotyrosine Western blot analysis indicates that both erbB-2 and erbB-3 are constitutively tyrosine phosphorylated in proliferating SUM-52PE cells. Conditioned medium obtained from SUM-52PE cells does not induce tyrosine phosphorylation of p185erbB-2 in a sensitive indicator cell line, suggesting that an erbB-2 activating factor is not secreted by these cells. However, neu differentiation factor/heregulin (NDF/HRG) mRNA is expressed by the cells, and Western blot analysis of SUM-52PE membrane protein revealed the presence of a M(r) 90,000 immunoreactive NDF/HRG protein. Thus, SUM-52PE cells synthesize a membrane bound form of NDF/HRG that may activate erbB-2 and erbB-3 via a juxtacrine mechanism. The addition of exogenous beta-2-NDF/HRG to the culture medium of SUM-52PE cells yields enhanced tyrosine phosphorylation of p185erbB-2/erbB-3 but has only a small stimulatory effect on the proliferation of these cells. By contrast, an erbB-2 monoclonal antibody that binds to the extracellular domain of erbB-2 is potently mitogenic for these cells. SUM-52PE cells were also found, by phosphotyrosine Western blot analysis, to express an inordinately large number of tyrosine phosphoproteins. Direct measurement of phosphotyrosine phosphatase (PTPase) activity in SUM-52PE cell membrane protein revealed 2-3-fold lower levels of PTPase activity compared to other normal and neoplastic breast epithelial cell lines. Thus, SUM-52PE cells exhibit altered growth phenotypes not identified previously in human breast cancer cells. The constitutive activation of erbB-2 and erbB-3 in these cells, coupled with their low, membrane-associated, PTPase activity are likely to play direct roles in driving proliferation of these breast cancer cells.
Ethier
S P
SP
Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor 48109-0582, USA.
Kokeny
K E
KE
Ridings
J W
JW
Dilts
C A
CA
D001483:Base Sequence; D015153:Blotting, Western; D001940:Breast; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D017931:DNA Primers; D066246:ErbB Receptors / Q000096:biosynthesis*; D005260:Female; D015870:Gene Expression; D018734:Genes, erbB-2; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D019000:Phosphotyrosine / Q000032:analysis; D010996:Pleural Effusion / Q000473:pathology; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis*; D020893:Receptor, ErbB-3; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D017931:DNA Primers; D066246:ErbB Receptors; D010750:Phosphoproteins; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D014638:Vanadates