Human Papillomavirus Type 16 E 6 and E 7 Cooperate to Increase Epidermal Growth Factor Receptor ( EGFR ) mRNA Levels , Overcoming Mechanisms by which Excessive EGFR Signaling Shortens the Life Span of Normal Human Keratinocytes 1 , 2

Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKc. These results prompted us to investigate the effects of overexpression of the EGFR in normal HKc. Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1–10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKc. In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKc. INTRODUCTION Oncogenic HPV is a major cause of cervical cancer and immortalizes HKc and cervical cells in culture (1–7). In our in vitro model system for HPV16-mediated carcinogenesis (3–5, 8), normal HKc transfected with HPV16 DNA undergo malignant transformation through a series of phenotypically well-defined “steps. ” Upon transfection with HPV16 DNA, normal HKc become immortalized, giving rise to stable lines (HKc/HPV16) that still retain growth requirements and differentiation properties similar to those of normal cells, and are not tumorigenic. Selection of HKc/HPV16 in serum-free MCDB153-LB medium devoid of EGF and BPE gives rise to EGFand BPE-independent lines (HKc/GFI). HKc/GFI are still responsive to differentiation stimuli (serum, high calcium) and are not tumorigenic. HKc/GFI can be selected further in medium supplemented with serum and high calcium (.0.3 m M) to yield differentiation-resistant lines (HKc/DR). HKc/DR, but not HKc/HPV16, are susceptible to malignant conversion by either vras or HSV2 sequences (9, 10). Previously we have shown that individual, independently derived HKc/HPV16 lines consistently exhibit higher EGF uptake and EGFR levels than normal HKc when cultured either in the presence or in the absence of exogenous EGF (8). The acquisition of the HKc/GFI phenotype is accompanied by an additional increase in EGFR protein levels. EGFR in HKc/GFI, but not HKc/HPV16 or normal HKc, are activated in the absence of EGF, and signaling through the EGFR is essential to maintain autonomous growth of HKc/GFI in the absence of EGF and BPE (8). The studies by Zyzak et al. (8) were conducted using well-established, immortal HKc/HPV16 lines in long-term culture and their respective HKc/GFI and HKc/DR derivatives. In the present study, we asked whether increases in EGFR can be detected early after the transfection of normal HKc with HPV16 DNA (which would exclude that long-term culturing is responsible for EGFR increases) and whether immortalization is necessary for this effect. In addition, we explore the possibility that acute expression of single HPV16 oncoproteins may produce an increase in EGFR in normal HKc. We show here that a robust increase in EGFR mRNA levels can be detected early after transfection of normal HKc with full-length HPV16 DNA, before the cells are immortalized. In addition, acute Received 5/8/00; accepted 3/1/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant RO1-CA62094 (to L. P.). 2 Presented in preliminary form at the 17th International Papillomavirus Conference in Charleston, SC, January 9–15, 1999. 3 To whom requests for reprints should be addressed, at Department of Pathology, University of South Carolina School of Medicine, Building 4, Room C-5, Columbia, SC 29208. Phone: (803) 733-3147; Fax: (803) 733-1515; E-mail: pirisi@med.sc. edu. 4 The abbreviations used are: HPV, human papillomavirus; HKc, human keratinocytes; HKc/HPV16, human keratinocytes immortalized with HPV16; EGF, epidermal growth factor; BPE, bovine pituitary extract; HKc/GFI, growth-factor independent human keratinocytes; HKc/DR, differentiation-resistant human keratinocytes; EGFR, epidermal rowth factor receptor; LB medium, Luria-Bertani medium; GFDM, growth factordepleted medium, lacking EGF and BPE; RPA, ribonuclease protection assay; ORF, open reading frame; PD, population doublings; Rb, Retinoblastoma Gene Product. 5 The unexpected bands of lower molecular weight produced by this EGFR probe with HKc/HPV16 and HKc/GFI mRNA (Fig. 1A) were mapped to two alternatively spliced EGFR mRNA forms. We have characterized these EGFR splice variants and found that their levels increase relative to the levels of wild-type EGFR mRNA in established HPV16-immortalized HKc but not in HKc acutely expressing HPV16 E6, E7, or E6 and E7 together (J. Liao, T. S. W. Engin, X. Xu, and L. Pirisi, manuscript in preparation).