Effects of cell cycle, wild-type p53 and DNA damage on p21^CIP1/Waf1 expression in human breast epithelial cells

In this study we examine the relationship between p21^CIP1/Waf1 (CIP1), a 21 kDa protein that binds to and modulates the activity of several cyclin dependent kinases and expression of wild-type (WT) p53 in human breast epithelial cells. Basal CIP1 protein, but not CIP1 mRNA levels correlated well with expression of WT p53 in human breast epithelial cells. To obtain more direct evidence that WT p53 regulated the level of CIP1 protein, the Human Papilloma Virus (HPV) E6 protein was introduced into immortalized 184B5 breast cells. Residual WT p53 levels correlated well with CIP1 protein but not CIP1 mRNA levels in isolated clones of transfected cells. CIP1 protein was increased at early times after growth factor arrested cells were stimulated to proliferate. The rise in CIP1 protein was due to a concomitant increase in CIP1 mRNA levels in MCF10, but not in normal mammary epithelial cells. DNA damage induced by ionizing radiation resulted in a transient increase in WT p53 levels but a prolonged induction of CIP1 protein. The sustained increase in CIP1 protein 24 h after radiation could not be attributed to a concomitant increase in CIP1 mRNA levels. Although the half-life of the CIP1 protein was not altered following irradiation, a fourfold increase in the amount of radioactivity incorporated into CIP1 protein was detected. When considered together these data suggest that wild-type p53 affects CIP1 protein accumulation at a posttranscriptional level in human breast epithelial cells under different physiologic and stress conditions.