Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation.
MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.
Ciardiello
F
F
Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892.
McGeady
M L
ML
Kim
N
N
Basolo
F
F
Hynes
N
N
Langton
B C
BC
Yokozaki
H
H
Saeki
T
T
Elliott
J W
JW
Masui
H
H
D015152:Blotting, Northern; D015153:Blotting, Western; D001940:Breast / Q000502:physiology*; D002455:Cell Division; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D066246:ErbB Receptors / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000276:immunology; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000235:genetics* / Q000276:immunology
D004247:DNA; D066246:ErbB Receptors; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D016211:Transforming Growth Factor alpha; C501009:HRAS protein, human