Cyclic nucleotide content of passaged keratinocytes in culture during various growth stages

Human newborn foreskin keratinocytes were maintained in culture in first passage for up to 60 days. An initial lag phase was followed by a growth and a final plateau phase. During this time, cell numbers, the morphologic appearance of both attached and detached cells, DNA, and cyclic nucleotide content were evaluated. Cell cycle analysis during growth phase was performed by flow cytofluorometry. High levels of cAMP and cGMP coincided with the early growth phase, when up to 70% of cells were in S, G2, or M compartments of the cell cycle. Halfway through growth phase, a fall in cyclic nucleotide levels coincided with a drop in mitotic index and increase in G1 cells. Plateau phase was characterized by increased desquamation, a gradual elevation of intracellular cAMP, cGMP, and DNA, and in later stages, an increased proportion of squamous cells in the attached layers. The oldest cultures were mostly squamous with low cyclic nucleotide levels. Results suggest that high cyclic nucleotide content is a feature of cycling, possibly S phase keratinocytes. Onset of differentiation coincides with a small but definite increase in both cAMP and cGMP, which in general vary in the same direction.