Stimulation of mitosis in cultured limb blastemata of the newt, Notophthalmus viridescens

Experiments were performed in vitro to assess the mitogenic activity of brain homogenates and cyclic nucleotides on adult newt limb blastemal cells. Early cone stage blastemata were explanted into organ culture, and thus denervated. The mitotic index (M.I.) of each was calculated and shown to decline steadily in vitro. The inclusion in the medium of newt brain homogenate (NBH) or dibutyryl cAMP (dbcAMP) delayed the loss of mitotic activity. Cyclic GMP alone or in combination with theophylline was ineffective as a mitogen. Bovine serum albumin and fetal calf serum were also ineffective, thus the mitogenic activity of brain homogenate appears to be distinct from a simple enrichment of the culture medium. Preliminary characterization of the active principles in NBH revealed a mitogenic activity that was both heat labile and trypsin sensitive. In related experiments, control cultures were maintained for 54 hours, roughly equivalent to one urodele blastemal cell cycle. When the decline in M. I. had stabilized, NBH or dbcAMP were added to the medium. A burst of mitotic activity was observed in both cases 8 hours later. Cyclic GMP was again ineffective. Since the G2 phase of the blastemal cell cycle lasts approximately four to eight hours, this result lends support to the hypothesis that blastemal cells are blocked in G2 in the absence of nerves. Cyclic AMP mimics the effect of brain homogenates at a specific phase of the blastemal cell cycle in vitro, and may therefore play some as yet undefined role in this neurotrophic process in vitro.