Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion.
Using an estrogen-inducible retroviral system, we demonstrate that oncogenic Raf-1 induces growth arrest and morphological changes in finite lifespan human mammary epithelial cells (HMEC). This arrest does not rely on expression of the cyclin-dependent kinase inhibitor (CKI) p16(INK4a), nor on changes in expression of the CKIs p21(Cip1), p14(ARF), p27(Kip1) or p57(Kip2). The Raf-induced arrest is independent of viral oncogene mediated inactivation of p53 and pRB, or c-myc overexpression. Flow cytometric analysis demonstrates that cells arrest in both G1 and G2. The Raf-induced arrest is mitigated or eliminated in some immortally transformed HMEC. Immortal HMEC that have both overcome replicative senescence and undergone the recently described conversion process maintain growth in the presence of transduced oncogenic Raf-1; they also gain EGF-independent growth and a low frequency of anchorage-independent growth. However, HMEC that have overcome replicative senescence but have not undergone conversion and HMEC immortalized by transduction with the catalytic subunit of telomerase, hTERT, remain severely growth arrested. These results indicate that the molecular mechanisms responsible for the Raf-1-induced growth arrest may vary among different finite lifespan cell types, and that in HMEC, this mechanism is altered during the conversion process, rather than as a direct consequence of overcoming senescence or expressing hTERT.
Olsen
Catherine L
CL
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, CA 94720, USA.
Gardie
Betty
B
Yaswen
Paul
P
Stampfer
Martha R
MR
D001940:Breast / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics*; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D005785:Gene Conversion; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D019098:Telomerase / Q000235:genetics*
D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D019908:Proto-Oncogene Proteins c-raf; D019098:Telomerase