Arizona Health Sciences

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations.

TitleImmortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations.
Publication TypeJournal Article
Year of Publication2014
AuthorsGarbe JC, Vrba L, Sputova K, Fuchs L, Novak P, Brothman AR, Jackson M, Chin K, LaBarge MA, Watts G, Futscher BW, Stampfer MR
JournalCell Cycle
Volume13
Issue21
Pagination3423-35
Date Published2014
ISSN1551-4005
KeywordsCell Aging, Cells, Cultured, Chromosome Aberrations, Cyclin-Dependent Kinase Inhibitor p16, Epigenesis, Genetic, Genomic Instability, Histones, Humans, Karyotyping, Mammary Glands, Human, Promoter Regions, Genetic, Proto-Oncogene Proteins c-myc, RNA Interference, RNA, Small Interfering, Telomerase
Abstract

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16(INK4); replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional "passenger" errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of "passenger" genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.

DOI10.4161/15384101.2014.954456
Alternate JournalCell Cycle
PubMed ID25485586
PubMed Central IDPMC4613853
Grant ListP30 CA023074 / CA / NCI NIH HHS / United States
R01 AG040081 / AG / NIA NIH HHS / United States
R00AG033176 / AG / NIA NIH HHS / United States
CA24844 / CA / NCI NIH HHS / United States
ES06694 / ES / NIEHS NIH HHS / United States
P30 ES006694 / ES / NIEHS NIH HHS / United States
CA23074 / CA / NCI NIH HHS / United States
R01AG040081 / AG / NIA NIH HHS / United States
Faculty Member Reference: 
George Watts, Ph.D.