Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells

James C. Garbe, Sanchita Bhattacharya, Batul Merchant, Ekaterina Bassett, Karen Swisshelm, Heidi Feiler, Andrew J. Wyrobek, Martha R. Stampfer

Research output: Contribution to journalArticle

103 Citations (Scopus)

Abstract

Normal human epithelial cells in culture have generally shown a limited proliferative potential of ∼10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence- associated β-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

Original languageEnglish (US)
Pages (from-to)7557-7568
Number of pages12
JournalCancer Research
Volume69
Issue number19
DOIs
StatePublished - Oct 1 2009
Externally publishedYes

Fingerprint

Telomere
Breast
Epithelial Cells
Population
Galactosidases
Cyclin-Dependent Kinase Inhibitor p16
Cyclin-Dependent Kinase Inhibitor p21
Retinoblastoma
Genomic Instability
Serum-Free Culture Media
Cell Lineage
DNA Damage
Cell Culture Techniques
Fibroblasts

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. / Garbe, James C.; Bhattacharya, Sanchita; Merchant, Batul; Bassett, Ekaterina; Swisshelm, Karen; Feiler, Heidi; Wyrobek, Andrew J.; Stampfer, Martha R.

In: Cancer Research, Vol. 69, No. 19, 01.10.2009, p. 7557-7568.

Research output: Contribution to journalArticle

Garbe, James C. ; Bhattacharya, Sanchita ; Merchant, Batul ; Bassett, Ekaterina ; Swisshelm, Karen ; Feiler, Heidi ; Wyrobek, Andrew J. ; Stampfer, Martha R. / Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. In: Cancer Research. 2009 ; Vol. 69, No. 19. pp. 7557-7568.
@article{0bf9900c34654ee7b9d0765867d315ee,
title = "Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells",
abstract = "Normal human epithelial cells in culture have generally shown a limited proliferative potential of ∼10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence- associated β-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.",
author = "Garbe, {James C.} and Sanchita Bhattacharya and Batul Merchant and Ekaterina Bassett and Karen Swisshelm and Heidi Feiler and Wyrobek, {Andrew J.} and Stampfer, {Martha R.}",
year = "2009",
month = "10",
day = "1",
doi = "10.1158/0008-5472.CAN-09-0270",
language = "English (US)",
volume = "69",
pages = "7557--7568",
journal = "Journal of Cancer Research",
issn = "0099-7013",
publisher = "American Association for Cancer Research Inc.",
number = "19",

}

TY - JOUR

T1 - Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells

AU - Garbe, James C.

AU - Bhattacharya, Sanchita

AU - Merchant, Batul

AU - Bassett, Ekaterina

AU - Swisshelm, Karen

AU - Feiler, Heidi

AU - Wyrobek, Andrew J.

AU - Stampfer, Martha R.

PY - 2009/10/1

Y1 - 2009/10/1

N2 - Normal human epithelial cells in culture have generally shown a limited proliferative potential of ∼10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence- associated β-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

AB - Normal human epithelial cells in culture have generally shown a limited proliferative potential of ∼10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence- associated β-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

UR - http://www.scopus.com/inward/record.url?scp=70350223685&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70350223685&partnerID=8YFLogxK

U2 - 10.1158/0008-5472.CAN-09-0270

DO - 10.1158/0008-5472.CAN-09-0270

M3 - Article

C2 - 19773443

AN - SCOPUS:70350223685

VL - 69

SP - 7557

EP - 7568

JO - Journal of Cancer Research

JF - Journal of Cancer Research

SN - 0099-7013

IS - 19

ER -