Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene.
The type III deletion-mutant gene for the epidermal growth factor receptor (EGFRvIII) is frequently expressed in glioblastomas and in breast and non-small cell lung carcinomas. To understand its contribution to the malignant phenotype in humans, we transfected NR6 cells with the mammalian vector pH beta APr-1-neo containing cDNA for either EGFRvIII or wild-type EGFR. Western blot analyses showed that NR6 transfected with wild-type EGFR (NR6W) contained a normal-sized protein (170 kilodaltons); cells transfected with EGFRvIII (NR6M) contained a truncated protein (145 kilodaltons). NR6W cells demonstrated a saturation binding curve with 125I-labeled EGF (affinity, 1.8 x 10(8); r2 = 0.96). NR6M cells, however, showed a low but consistent level of 125I-labeled EGF binding (affinity, 4 x 10(7); r2 = 0.99) compared with NR6, which lacked binding. The population doubling time was shorter for NR6M (0.64 days) than for NR6W (1.1 days) and NR6V (2.27 days). Soft agar focus formation assay by NR6M was 4- to 5-fold higher than that by NR6W. In nude mice, NR6M (1 x 10(7) cells), without exogenous ligand, formed tumors within 12 days; no tumors were observed over 90 days in mice receiving identical doses of NR6W, NR6V, or NR6 cells. EGF stimulated autophosphorylation of receptor in NR6W (4- to 9-fold) but caused only slight (1.8- to 1.9-fold) to no enhancement in NR6M. Further, there was no difference in constitutive tyrosine kinase activity between NR6M and NR6W. Our results clearly indicate that EGFRvIII functions as an oncoprotein, but its intrinsic tyrosine kinase activity may not be responsible for its biological function.
Batra
S K
SK
Duke University Medical Center, Durham, North Carolina 27710, USA.
Castelino-Prabhu
S
S
Wikstrand
C J
CJ
Zhu
X
X
Humphrey
P A
PA
Friedman
H S
HS
Bigner
D D
DD
D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D015513:Oncogene Proteins / Q000502:physiology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D017384:Sequence Deletion; D014162:Transfection
D015513:Oncogene Proteins; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases