DNA Synthesis in Mouse Epidermis: S Phase Cells that Remain Unlabeled After Pulse Labeling with DNA Precursors Progress Slowly Through S

Epidermal basal cells from hairless mice were isolated after pulse labeling with tritiated DNA precursors and subjected to DNA flow cytometry combined with cell sorting. Cells were sorted from a window in the middle of the S phase, collected on glass slides, and subjected to autoradiography. Unlabeled cells in the middle of the S phase were found in normal mouse epidermis after optimal pulse labeling with tritiated thymidine ([3H]dThd), in accordance with previous results. The proportion of unlabeled S phase cells was considerably increased among basal cells from mice treated with growth-inhibitory epidermal extracts. Reanalysis and re-sorting of cells previously sorted from mid S showed that unlabeled cells could not be accounted for by G1 contamination. Furthermore, labeling with precursors incorporated into DNA by “de novo” metabolic pathway ([3H]Urd) did not reduce the proportion of unlabeled S phase cells, either when given alone or when given in combination with the precursor for DNA incorporated by the “salvage” pathway ([3H]dThd). This strongly indicates that the unlabeled S phase cells do not synthesize DNA continuously, or are synthesizing DNA at a rate below the level of detection. A reduced proportion of unlabeled S phase cells was found in regenerating epidermis. This may be explained by a dilution effect caused by the 3-fold increase in the total number of cells within S phase at this condition. The observation that essentially all cells in mid S phase were labeled during 4 days of continuous labeling with [3H]dThd, indicates that cells in S phase that remain unlabeled after optimal pulse labeling are cycling, albeit slowly. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells are larger than the average. These cells may represent a subpopulation of basal cells going through their last division cycle before differentiation.