Effects of methotrexate in vitro on epidermal cell proliferation

Epidermal cell migratory activity and epidermal cell proliferation are clearly dissociable in explant culture. Epidermal cell migration requires a non-dialysable, 65,000 mol.wt factor which is destroyed at 100 C but is stable at 80 C for at least 30 min. In the presence of dialysed serum or heated serum (80° C for 30 min) or DNA synthesis inhibitors (methotrexate or hydroxyurea), cells will migrate from the explant but will not proliferate. At least two factors are required for normal proliferation under these restricted conditions—an adequate supply of DNA precursers, i.e. nucleosides, and a heat labile (80° C) non-dialysable serum component. Methotrexate in concentrations of 1.0 μg/ml or greater added to cultures in normal fetal calf serum significantly inhibited mitoses; however, when added to serum dialysed to remove thymidine, mitotic inhibition occurred at a concentration of 0.1 μg/ml of methotrexate and when added to dialysed serum and kept in dialysed serum, inhibition occurred with 0.01 μg/ml of methotrexate. Methotrexate did not inhibit outgrowth. Hydroxyurea also inhibited mitoses but did not effect outgrowth.