title: Increased levels and constitutive tyrosine phosphorylation of the epidermal growth factor receptor contribute to autonomous growth of human papillomavirus type 16 immortalized human keratinocytes.
abstract: Transfection of individual normal human foreskin keratinocyte (HKc) strains with human papillomavirus type 16 (HPV16) DNA results in the establishment of immortalized cell lines (HKc/HPV16) which, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation in serum-free media. However, sublines which proliferate in serum-free media in the absence of EGF and BPE can be reproducibly established from individual HKc/HPV16 lines, following selection in serum-free media lacking EGF and BPE. The growth factor-independent sublines (HKc/GFI) proliferate in the absence of EGF and BPE at the same rate and to the same extent as in medium supplemented with these growth factors, whereas the parental HKc/HPV16 lines proliferate poorly in the absence of EGF and BPE. As a first approach to understanding the molecular basis by which HKc/GFI have lost their requirement for EGF, we compared EGF uptake and EGF receptor (EGFR) numbers in normal HKc, HKc/HPV16, and HKc/GFI. HKc/GFI exhibit increased EGF uptake and increased EGFR numbers compared to HKc/HPV16. A neutralizing antibody against the extracellular domain of the EGFR dramatically inhibited clonal growth of HKc/GFI, indicating that signaling through the EGFR must be important for the ability of HKc/GFI to proliferate in the absence of EGF. In addition, while in the absence of EGF normal HKc and HKc/HPV16 exhibited no detectable EGFR tyrosine phosphorylation, the EGFRs in HKc/GFI were tyrosine phosphorylated in the absence of EGF and hyperphosphorylated in the presence of EGF. Although an anti-TGF-alpha antibody inhibited the growth of HKc/GFI, we unexpectedly found that HKc/GFI and HKc/HPV16 secreted comparable and extremely low amounts of TGF-alpha (4 to 9 pg/10(6) cells per 24 h); about 100- to 250-fold less than normal HKc (1018 pg/10(6) cells per 24 h). No other ligands for the EGFR were detected in media conditioned by normal HKc, HKc/HPV16, or HKc/GFI. Thus, while overexpression and constitutive activation of the EGFR appear to be important features of HKc/GFI, enhanced secretion of TGF-alpha or other ligands for the EGFR does not explain the proliferation of HKc/GFI in the absence of EGF and BPE.
authors.affiliation: Department of Pediatrics, University of South Carolina School of Medicine, Columbia.
authors.forename: L L
authors.initials: LL
authors.lastname: Zyzak
authors.forename: L M
authors.initials: LM
authors.lastname: MacDonald
authors.forename: A
authors.initials: A
authors.lastname: Batova
authors.forename: R
authors.initials: R
authors.lastname: Forand
authors.forename: K E
authors.initials: KE
authors.lastname: Creek
authors.forename: L
authors.initials: L
authors.lastname: Pirisi
mesh_terms: D000911:Antibodies, Monoclonal; D002455:Cell Division; D002461:Cell Line, Transformed; D017077:Culture Media, Conditioned / Q000737:chemistry; D016895:Culture Media, Serum-Free; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D009500:Neutralization Tests; D027383:Papillomaviridae; D010766:Phosphorylation; D012867:Skin / Q000166:cytology; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016211:Transforming Growth Factor alpha; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors