Episomally mediated overexpression of wild-type erbB-2 transforms MCF-10A breast epithelial cells.
MCF-10A are human non-transformed, EGF and insulin dependent breast epithelial cells. The cells were transfected with an episomal pCEP4 vector library containing cDNA from SKBR3 breast carcinoma cells, and selected in media without EGF. After two cycles of expression cloning, morphologically transformed cells appeared. Extracted episomes contained a high proportion of erbB-2 cDNA with wild-type transmembrane domains. Transfection of MCF-10A with individual erbB-2 containing episomes induced significant foci formation in low serum (0.1%) without EGF. MCF-10A sublines expanded from these foci contained a high number of erbB-2 gene copies, highly expressed erbB-2, and lost E-cadherin expression. These results show that if wild-type erbB-2 is sufficiently overexpressed, erbB-2 alone can cause EGF independent transformation of these nonmalignant breast cells. This expression system may be useful for expression cloning in MCF-10A cells, and simulating the effects of high erbB-2 gene amplification in breast epithelial cells.
Yu
Bennett W
BW
Oncology Lab, Division of Hematology and Oncology, Josephine Ford Cancer Center, Henry Ford Health System, Detroit, MI, USA. yub@karmanos.org
Bowers
Mark
M
Witkowski
Greg
G
Huang
Meiyi
M
Ramachandran
Satyabama
S
D001940:Breast / Q000473:pathology*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002461:Cell Line, Transformed / Q000473:pathology; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000473:pathology; D002999:Clone Cells / Q000473:pathology; D004274:DNA, Recombinant / Q000235:genetics; D004815:Epidermal Growth Factor / Q000502:physiology; D004847:Epithelial Cells / Q000473:pathology*; D005784:Gene Amplification / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D018734:Genes, erbB-2 / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D014162:Transfection
D004274:DNA, Recombinant; D004815:Epidermal Growth Factor