TRANSFORMING GROWTH-FACTOR-BETA-1 SUPPORTS AUTONOMOUS GROWTH OF HUMANPAPILLOMAVIRUS-IMMORTALIZED CERVICAL KERATINOCYTES UNDER CONDITIONS PROMOTING SQUAMOUS DIFFERENTIATION

Transforming growth factor beta (TGF-beta) inhibits proliferation of k eratinocytes cultured from normal anogenital epithelia; however, human papillomavirus (HPV)-immortalized cell lines often exhibit increased resistance. Present results demonstrate that TGF-beta 1 (1-10 pM) stim ulates growth of multiple HPV-immortalized cell lines when cultures ar e maintained under conditions promoting squamous differentiation (MCDB 153-LB medium with 1.0 mM calcium and without epidermal growth factor and bovine pituitary extract). Growth stimulation by TGF-beta 1 was no t due to altered expression of type I or II receptors, but was increas ed after extended passage of cells in culture. Differentiation of immo rtal keratinocytes resulted in induction of RNAs encoding two markers of squamous differentiation, involucrin and keratin 1, and decreased e xpression of RNAs for the epidermal growth factor (EGF) receptor and t wo ligands, amphiregulin and TGF-alpha. Growth stimulation by TGF-beta 1 occurred indirectly via establishment of an autocrine loop, TGF-bet a 1 increased expression of RNAs encoding the EGF-R and amphiregulin, and also increased numbers of cell-surface EGF-Rs without altering the ir affinity. In contrast, TGF-beta 1 inhibited autonomous growth and t ranscription of amphiregulin RNA in normal keratinocytes. Growth stimu lation by TGF-beta 1 could be blocked by a monoclonal antibody that co mpetes for binding to the EGF-R or by a mixture of monoclonal antibodi es that neutralize amphiregulin activity, confirming the importance of this autocrine pathway, Thus, partial abrogation of the growth inhibi tory response to TGF-beta 1 sensitizes HPV-immortalized keratinocytes to a growth stimulatory signal mediated by an EGF-R-dependent pathway involving autocrine stimulation by amphiregulin.