p57(KIP2) expression and loss heterozygosity during immortal conversion ofcultured human mammary epithelial cells

We have uncovered a novel role for the cyclin-dependent kinase inhibitor, p 57(KIP2), during the immortalization of cultured human mammary epithelial c ells (HMECs). HMECs immortalized after chemical carcinogen exposure initial ly expressed little or no telomerase activity, and their telomeres continue d to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined to less than or equal to 3 kb exhibited slo w heterogeneous growth and contained many nonproliferative cells. These con ditionally immortal HMEC cultures accumulated large quantities of p57 prote in. With continued passage, the conditionally immortal cell populations ver y gradually converted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerase activity, and stabilization of tel omere length. The fully immortal HMECs that grew well did not accumulate p5 7 in G(o) or during the cell cycle. DNA and RNA analysis of mass population s and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically shor t telomeres was repeatedly accompanied by loss of the expressed p57 allele and transient expression of the allele imprinted previously. Conditionally immortal 184A1 with mean TRF >3 kb, infected with retroviruses containing t he p57 gene, exhibited premature slow heterogeneous growth. Conversely, exo genous expression of human telomerase reverse transcriptase (hTERT), the ca talytic subunit of telomerase, in 184A1 with mean TRF >3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling popu lations. These data indicate that in HMECs that have overcome replicative s enescence, p57 may provide an additional barrier against indefinite prolife ration. Overcoming p57-mediated growth inhibition in these cells may be cru cial for acquisition of the unlimited growth potential thought to be critic al for malignant progression.