Two subtypes of reversible cell cycle restriction points exist in cultured normal human keratinocyte progenitor cells

Three methods can induce reversible arrest of the growth of cultured human keratinocytes in the G1 phase of the cell cycle. These include the incubation of cells in medium containing transforming growth factor (TGF)-beta or ethionine, or in isoleucine-deficient medium. The current studies were performed to determine if the growth arrest induced by these methods occurs at a common or at a distinct G1 state(s). We first evaluated the relative time interval required for arrested cells to initiate DNA synthesis after growth restimulation with mitogenic medium. The results show that ethionine arrested cells require 22 to 28 hours to initiate DNA synthesis and that a maximum rate of DNA synthesis occurs at 46 hours. Cells arrested by isoleucine deficiency required 10 to 12 hours to initiate DNA synthesis with peak DNA synthesis occurring at 24 hours. Finally, TGF-beta arrested cells require only 6 to 8 hours to initiate DNA synthesis and show a maximum rate of DNA synthesis at 18 hours. We next evaluated if cells that were growth arrested at these states were differentially capable of initiating DNA synthesis in different types of potentially mitogenic medium. The results show that if TGF-beta arrested cells were refed TGB-beta free serum containing medium with ethionine or similar medium with isoleucine deficiency, no DNA synthesis occurred. In contrast, if cells whose growth was arrested in ethionine-containing medium or in isoleucine-deficient medium were refed mitogenic medium containing TGF-beta, significant DNA synthesis was detected. These results suggest that least two different types of reversible growth arrest states exist in cultured human keratinocytes. One appears to be mediated by receptor-dependent processes, such as that induced by TGF-beta, and the other appears to be mediated by other types of metabolic events, such as those induced by ethionine treatment or by isoleucine deficiency.