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

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 p16INK4; 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,more » 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.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [2] ;  [4] ;  [5] ;  [6] ;  [1] ;  [2] ;  [2] ;  [7]
  1. Lawrence Berkeley National Laboratory, Berkeley, California, (United States), Life Sciences Division
  2. Arizona Cancer Center; The University of Arizona; Tucson, AZ USA
  3. Department of Pathology; The University of Arizona College of Medicine; Tucson, AZ USA
  4. Department of Pathology; The University of Arizona College of Medicine; Tucson, AZ USA;
  5. Case Comprehensive Cancer Center; Case Western Reserve University; Cleveland, OH USA
  6. 7 University of California San Francisco; San Francisco, CA USA
  7. Lawrence Berkeley National Laboratory, Berkeley, California, (United States), Life Sciences Division; 2 Arizona Cancer Center; The University of Arizona; Tucson, AZ USA
Publication Date:
OSTI Identifier:
1220569
Type:
Accepted Manuscript
Journal Name:
Cell Cycle (Georgetown, Tex)
Additional Journal Information:
Journal Name: Cell Cycle (Georgetown, Tex); Journal Volume: 13; Journal Issue: 21; Journal ID: ISSN 1538-4101
Publisher:
Taylor and Francis
Research Org:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES carcinogenesis; c-Myc; genomic instability; human mammary epithelial cells; immortalization; p16INK4a; senescence; telomerase