title: Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations.
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.
authors.affiliation: a Life Sciences Division ; Lawrence Berkeley National Laboratory ; Berkeley , CA USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Garbe
authors.forename: Lukas
authors.initials: L
authors.lastname: Vrba
authors.forename: Klara
authors.initials: K
authors.lastname: Sputova
authors.forename: Laura
authors.initials: L
authors.lastname: Fuchs
authors.forename: Petr
authors.initials: P
authors.lastname: Novak
authors.forename: Arthur R
authors.initials: AR
authors.lastname: Brothman
authors.forename: Mark
authors.initials: M
authors.lastname: Jackson
authors.forename: Koei
authors.initials: K
authors.lastname: Chin
authors.forename: Mark A
authors.initials: MA
authors.lastname: LaBarge
authors.forename: George
authors.initials: G
authors.lastname: Watts
authors.forename: Bernard W
authors.initials: BW
authors.lastname: Futscher
authors.forename: Martha R
authors.initials: MR
authors.lastname: Stampfer
mesh_terms: D002478:Cells, Cultured; D016922:Cellular Senescence; D002869:Chromosome Aberrations; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic; D042822:Genomic Instability; D006657:Histones / Q000378:metabolism; D006801:Humans; D007621:Karyotyping; D042361:Mammary Glands, Human / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D006657:Histones; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D019098:Telomerase
keywords: BaP, benzo(a)pyrene; CT, cholera toxin; DDR, DNA damage response; DMR, differentially methylated regions; HMEC, human mammary epithelial cells; OIS, oncogene-induced senescence; PD, population doublings; RB, retinoblastoma protein; TTS, transcription start site; X, oxytocin; c-Myc; carcinogenesis; genomic instability; human mammary epithelial cells; immortalization; p, passage; p16INK4a; p16sh, shRNA to p16INK4A; senescence; telomerase