Transforming growth factor beta 1 supports autonomous growth of human papillomavirus-immortalized cervical keratinocytes under conditions promoting squamous differentiation

Transforming growth factor fi (TGF-�) inhibits proliferation of keratinocytes cultured from normal anogenital epithelia; however, human papillomavirus (HPV)-immortalized cell lines often exhibit increased resistance. Present results demonstrate that TGF-fil (1-10 pM) stimulates growth of multiple HPVimmortalized cell lines when cultures are maintained under conditions promoting squamous differentiation (MCDBI53-LB medium with 1.0 mp.icalcium and without epidermal growth factor and bovine pituitary extract). Growth stimulation by TGF-fil was not due to altered expression of type I or II receptors, but was increased after extended passage of cells in culture. Differentiation of immortal keratinocytes resulted in induction of RNAS encoding two markers of squamous differentiation, involucrin and keratin I , and decreased expression of RNAs for the epidermal growth factor (EGF) receptor and two ligands, amphiregulin and TGFa. Growth stimulation by TGF-fil occurred indirectly via establishment of an autocrine loop. TGF-fil increased expression of RNAS encoding the EGF-R and amphiregulin, and also increased numbers of cellsurface EGF-Rs without altering their affinity. In contrast, TGF-f31 inhibited autonomous growth and transcription of amphiregulin RNA in normal keratinocytes. Growth stimulation by TGF-fil could be blocked by a monoclonal antibody that competes for binding to the EGF-R or by a mixture of monoclonal antibodies that neutralize amphiregulin activity, confirming the importance of this autocnne pathway. Thus, partial abrogation of the growth inhibitory response to TGF-fil sensitizes HPV-immortalized