Expression of wild-type p53 during the cell cycle in normal human mammary epithelial cells

In this study, we compare the expression patterns of p53 mRNA and protein in normal human mammary epithelial cells following synchronization to different points in the cell cycle using two independent methods. When treated with lovastatin, the cells were blocked in G1 and appeared to express increased levels of wild-type p53 when examined by immunostaining. Upon reversal of the metabolic block, the number of nuclei that stained positively for p53 declined dramatically during mid-G1 and increased again concomitant with the entry of cells into S phase. In contrast to the immunostaining results, Northern and Western blot analyses revealed little change in p53 mRNA and protein levels in the lovastatin-synchronized cells. When normal human mammary epithelial cells were made quiescent by removal of growth factors, the mRNA for p53 showed a biphasic distribution. p53 mRNA levels were increased during growth arrest, decreased during the G1 phase, and rose again concomitant with the entry of cells into S phase. The immunostaining pattern of p53 also showed a biphasic distribution similar to the pattern of mRNA expression. Despite an increase in p53 mRNA and immunostaining levels, growth factor-arrested cells actually had less total p53 protein. Upon stimulation to proliferate, p53 protein levels remained low throughout G1 and increased concomitant with the entry of cells into S phase. Taken together, the results from these studies demonstrate that p53 immunostaining patterns do not correlate with the overall levels of p53 protein at different times during the cell cycle. Therefore, the distinct changes observed in p53 immunostaining patterns are likely due to posttranslational modifications, conformational changes, or interactions of p53 with other cellular proteins during the cell cycle.