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.