Abstract
BackgroundShortening of telomeres, which are essential for maintenance of genomic integrity, is a mechanism commonly associated with the aging process. Here we ascertained whether changes in telomere lengths or telomerase activity correlated with age in normal human mammary epithelial cells (HMEC), or with phenotypes of aging in breast. Accordingly, flow cytometry fluorescence in situ hybridization (flowFISH) was used to determine relative telomere lengths (RTL), and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP), in a collection of 41 primary HMEC strains established from women aged 16 to 91 years.ResultsRTL measurements of HMEC strains that were heterogeneous with respect to lineage composition revealed no significant associations between telomere length with age, maximum observed population doublings, or with lineage composition of the strains. However, within strains, luminal epithelial and cKit-expressing epithelial progenitor cells that were flow cytometry-enriched from individual HMEC strains exhibited significantly shorter telomeres relative to isogenic myoepithelial cells (P < 0.01). In unsorted strains, detectable telomerase activity did not correlate with RTL. Telomerase activity declined with age; the average age of strains that exhibited TRAP activity was 29.7 ± 3.9y, whereas the average age of strains with no detectable TRAP activity was 49.0 ± 4.9y (P < 0.01). Non-detectable TRAP activity also was correlated with phenotypes of aging previously described in HMEC strains; increased proportions of CD227-expressing luminal epithelial cells (P < 0.05) and cKit-expressing progenitor cells (P < 0.05).ConclusionsTelomere shortening did not correlate with the chronological ages of HMEC strains, whereas decreased telomerase activity correlated with age and with lineage distribution phenotypes characteristic of aging.
Highlights
Shortening of telomeres, which are essential for maintenance of genomic integrity, is a mechanism commonly associated with the aging process
Because telomere shortening has been associated with aging and short stable telomeres are a characteristic of carcinomas we considered that age-dependent shortening of telomeres in human mammary epithelial cells (HMEC) could contribute to the observed age-associated changes
Relative telomere lengths (RTL) measured with flow cytometry-based fluorescence in situ hybridization were validated by comparing relative telomere lengths (RTL) to telomere lengths previously determined by mean telomere restriction fragment (TRF) Southern blots in a wellcharacterized normal HMEC strain and an immortal cell line derivative [21,22]
Summary
Shortening of telomeres, which are essential for maintenance of genomic integrity, is a mechanism commonly associated with the aging process. We ascertained whether changes in telomere lengths or telomerase activity correlated with age in normal human mammary epithelial cells (HMEC), or with phenotypes of aging in breast. We have generated a large collection of cultured normal human mammary epithelial cells (HMEC) strains to better understand whether the aging process alters, the mammary epithelia in potentially deleterious ways [1]. This resource consists of multiple strains of normal finite lifespan pre-stasis HMEC established from discarded reduction mammoplasty and peripheral to tumour mastectomy tissues from women aged 16 to 91 years. Because telomere shortening has been associated with aging and short stable telomeres are a characteristic of carcinomas we considered that age-dependent shortening of telomeres in HMEC could contribute to the observed age-associated changes
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