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

James C. Garbe, Lukas Vrba, Klara Sputova, Laura Fuchs, Petr Novak, Arthur R. Brothman, Mark Jackson, Kwang-Yung Chin, Mark A. La Barge, George Watts, Bernard W. Futscher, Martha R. Stampfer

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 nonclonal 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, 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.

Original languageEnglish (US)
Pages (from-to)3423-3435
Number of pages13
JournalCell Cycle
Volume13
Issue number21
DOIs
StatePublished - Nov 1 2014
Externally publishedYes

Fingerprint

Breast
Epithelial Cells
Telomerase
Telomere Shortening
Carcinoma
Genomic Instability
Cell Aging
Telomere
Karyotype
Small Interfering RNA
Neoplasms

Keywords

  • C-Myc
  • Carcinogenesis
  • Genomic instability
  • Human mammary epithelial cells
  • Immortalization
  • P16INK4a
  • Senescence
  • Telomerase

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Developmental Biology

Cite this

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations. / Garbe, James C.; Vrba, Lukas; Sputova, Klara; Fuchs, Laura; Novak, Petr; Brothman, Arthur R.; Jackson, Mark; Chin, Kwang-Yung; La Barge, Mark A.; Watts, George; Futscher, Bernard W.; Stampfer, Martha R.

In: Cell Cycle, Vol. 13, No. 21, 01.11.2014, p. 3423-3435.

Research output: Contribution to journalArticle

Garbe, JC, Vrba, L, Sputova, K, Fuchs, L, Novak, P, Brothman, AR, Jackson, M, Chin, K-Y, La Barge, MA, Watts, G, Futscher, BW & Stampfer, MR 2014, 'Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations', Cell Cycle, vol. 13, no. 21, pp. 3423-3435. https://doi.org/10.4161/15384101.2014.954456
Garbe, James C. ; Vrba, Lukas ; Sputova, Klara ; Fuchs, Laura ; Novak, Petr ; Brothman, Arthur R. ; Jackson, Mark ; Chin, Kwang-Yung ; La Barge, Mark A. ; Watts, George ; Futscher, Bernard W. ; Stampfer, Martha R. / Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations. In: Cell Cycle. 2014 ; Vol. 13, No. 21. pp. 3423-3435.
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