Human Mammary Epithelial Cells (HMECs) grown in serum-free medium exhibit two phases of growth, before they senesce in culture. The first growth phase, referred to as pre-selection, is short and followed by a temporary growth arrest where epigenetic changes can occur to give rise to post-selection cells with altered growth properties. Post-selection HMECs may represent a transit amplifying stem cell population because they exhibit moderate levels of the stem cell marker, ALDH and can produce mammospheres with disorganised lumen through-out their growth phase. On the other hand, pre-selection HMECs retain a small bipotent stem cell population producing mammospheres with a discrete lumen at early passages.
Post-selection cells are characterised by loss of p16 INK4a expression and despite their increased growth rate, they exhibit elevated levels of p53, p21WAF1/CIP1 and Mdm2, but can still initiate a normal p53 response to DNA damage. We found that the post-selection HMECs contain a truncated Mdm2 protein, p60, which lacks the ubiquitylation domain and propose that the increased levels of p53 in post-selection HMECs are due to the presence of an Mdm2 fragment that binds to but does not result in its degradation. The observation, that nutlin-3a treatment did not alter the levels of p53 in post-selection cells, supports our conclusion that the majority of the p53 in these cells is not susceptible to Mdm2-mediated degradation. Exogenous expression of p16 INK4a in post-selection cells decreased levels of both p53 transcript and protein and this effect was inhibited by nutlin-3a indicating that p16 INK4a can regulate p53 expression by affecting both p53 transcription and mdm2-dependent degradation of p53. Thus we propose that post-selection HMECs are able to proliferate rapidly in the presence of elevated levels of p53 and initiate a normal stress response because they harbour a truncated form of Mdm2 p60.