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Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length.

Sputova K, Garbe JC, Pelissier FA, Chang E, Stampfer MR, Labarge MA - Genome Integr (2013)

Bottom Line: 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.Telomere 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. MALabarge@lbl.gov.

ABSTRACT

Background: Shortening 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.

Results: RTL 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).

Conclusions: Telomere 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.

No MeSH data available.


Related in: MedlinePlus

Relative telomere length and telomerase activity varies by lineage within cultured strains. (A) Representative FACS analyses of CD227 and CD10 expression in 4th passage HMEC strains isolated from a 19 year old woman (240L) and a 91 year old woman (805P). FACS plots are shown as dot plots, at left are isotype antibody controls, and at right are the CD10 and CD227 stained samples. Luminal and myoepithelial lineages are identified as LEP and MEP, respectively. (B) Histogram of CD117 expression by flow cytometry analysis of isotope negative control staining (black lines) and CD117 expression (blue lines) from 4th passage HMEC strains 240L and 805P. Histograms of ratios of keratin (K)14 to K19 protein expression in FACS enriched lineages, MEP (CD117-/CD227-/CD10+), LEP (CD117-/CD227+/CD10-), cKit + (CD117+) and unsorted cells from 4th passage strains (C) 240L and (D) 805P (n = 500 cells/histogram). Insets show examples of FACS enriched cells stained to show K14 (red), K19 (green), and nuclei (blue). (E) RTL measurements in FACS enriched lineages strains 240L and 805P at 4th passage, (n = 4). * = P < 0.05, ** = P < 0.01, ***P < 0.001.
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Figure 2: Relative telomere length and telomerase activity varies by lineage within cultured strains. (A) Representative FACS analyses of CD227 and CD10 expression in 4th passage HMEC strains isolated from a 19 year old woman (240L) and a 91 year old woman (805P). FACS plots are shown as dot plots, at left are isotype antibody controls, and at right are the CD10 and CD227 stained samples. Luminal and myoepithelial lineages are identified as LEP and MEP, respectively. (B) Histogram of CD117 expression by flow cytometry analysis of isotope negative control staining (black lines) and CD117 expression (blue lines) from 4th passage HMEC strains 240L and 805P. Histograms of ratios of keratin (K)14 to K19 protein expression in FACS enriched lineages, MEP (CD117-/CD227-/CD10+), LEP (CD117-/CD227+/CD10-), cKit + (CD117+) and unsorted cells from 4th passage strains (C) 240L and (D) 805P (n = 500 cells/histogram). Insets show examples of FACS enriched cells stained to show K14 (red), K19 (green), and nuclei (blue). (E) RTL measurements in FACS enriched lineages strains 240L and 805P at 4th passage, (n = 4). * = P < 0.05, ** = P < 0.01, ***P < 0.001.

Mentions: To determine whether FACS-based RTL measurements would reveal that telomere length varied as a function of lineage in HMEC, as was previously reported from quantitative (Q)-FISH measurements in breast tissue sections [19,20], RTL were measured in LEP, MEP (Figure 2A), and cKit + lineages (Figure 2B) that were FACS-enriched from p4 HMEC strains derived from a 19y woman (strain 240L) and a 91y woman (strain 805P). Strain 805P had a larger proportion of CD227+ LEP and cKit+ progenitor HMEC compared to the younger 240L strain, as we predicted based on their relative ages. Enrichment of the three lineages was verified by automated image analysis of immunofluorescent staining for intermediate filament protein keratin (K)14 and K19 expression in sorted HMEC. Consistent with our previously reported observations of age-dependent lineage phenotypes, analysis confirmed that in HMEC from the 19y woman the MEP were enriched for the K14+/K19- phenotype, LEP for the K14-/K19+ phenotype, and cKit progenitors for the K14+/K19+ phenotype (Figure 2C), whereas in the 91y woman all lineages were unusually enriched for K14 expression consistent with previous observations [1] (Figure 2D). Within both strains, the RTL significantly differed by lineage, where both LEP and cKit + cells exhibited shorter telomeres than the MEP (Figure 2E). Although significant, the differences in RTL between the lineages of the two strains were less than 1.5-fold, and the measurements of mixed populations, as in Figure 1B, essentially represent average RTL. If anything the average RTL of older strains would be biased in the direction of shortened telomere lengths, thus making our conclusion that HMEC telomeres are unlikely to shorten significantly with age all the more strongly.


Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length.

Sputova K, Garbe JC, Pelissier FA, Chang E, Stampfer MR, Labarge MA - Genome Integr (2013)

Relative telomere length and telomerase activity varies by lineage within cultured strains. (A) Representative FACS analyses of CD227 and CD10 expression in 4th passage HMEC strains isolated from a 19 year old woman (240L) and a 91 year old woman (805P). FACS plots are shown as dot plots, at left are isotype antibody controls, and at right are the CD10 and CD227 stained samples. Luminal and myoepithelial lineages are identified as LEP and MEP, respectively. (B) Histogram of CD117 expression by flow cytometry analysis of isotope negative control staining (black lines) and CD117 expression (blue lines) from 4th passage HMEC strains 240L and 805P. Histograms of ratios of keratin (K)14 to K19 protein expression in FACS enriched lineages, MEP (CD117-/CD227-/CD10+), LEP (CD117-/CD227+/CD10-), cKit + (CD117+) and unsorted cells from 4th passage strains (C) 240L and (D) 805P (n = 500 cells/histogram). Insets show examples of FACS enriched cells stained to show K14 (red), K19 (green), and nuclei (blue). (E) RTL measurements in FACS enriched lineages strains 240L and 805P at 4th passage, (n = 4). * = P < 0.05, ** = P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Relative telomere length and telomerase activity varies by lineage within cultured strains. (A) Representative FACS analyses of CD227 and CD10 expression in 4th passage HMEC strains isolated from a 19 year old woman (240L) and a 91 year old woman (805P). FACS plots are shown as dot plots, at left are isotype antibody controls, and at right are the CD10 and CD227 stained samples. Luminal and myoepithelial lineages are identified as LEP and MEP, respectively. (B) Histogram of CD117 expression by flow cytometry analysis of isotope negative control staining (black lines) and CD117 expression (blue lines) from 4th passage HMEC strains 240L and 805P. Histograms of ratios of keratin (K)14 to K19 protein expression in FACS enriched lineages, MEP (CD117-/CD227-/CD10+), LEP (CD117-/CD227+/CD10-), cKit + (CD117+) and unsorted cells from 4th passage strains (C) 240L and (D) 805P (n = 500 cells/histogram). Insets show examples of FACS enriched cells stained to show K14 (red), K19 (green), and nuclei (blue). (E) RTL measurements in FACS enriched lineages strains 240L and 805P at 4th passage, (n = 4). * = P < 0.05, ** = P < 0.01, ***P < 0.001.
Mentions: To determine whether FACS-based RTL measurements would reveal that telomere length varied as a function of lineage in HMEC, as was previously reported from quantitative (Q)-FISH measurements in breast tissue sections [19,20], RTL were measured in LEP, MEP (Figure 2A), and cKit + lineages (Figure 2B) that were FACS-enriched from p4 HMEC strains derived from a 19y woman (strain 240L) and a 91y woman (strain 805P). Strain 805P had a larger proportion of CD227+ LEP and cKit+ progenitor HMEC compared to the younger 240L strain, as we predicted based on their relative ages. Enrichment of the three lineages was verified by automated image analysis of immunofluorescent staining for intermediate filament protein keratin (K)14 and K19 expression in sorted HMEC. Consistent with our previously reported observations of age-dependent lineage phenotypes, analysis confirmed that in HMEC from the 19y woman the MEP were enriched for the K14+/K19- phenotype, LEP for the K14-/K19+ phenotype, and cKit progenitors for the K14+/K19+ phenotype (Figure 2C), whereas in the 91y woman all lineages were unusually enriched for K14 expression consistent with previous observations [1] (Figure 2D). Within both strains, the RTL significantly differed by lineage, where both LEP and cKit + cells exhibited shorter telomeres than the MEP (Figure 2E). Although significant, the differences in RTL between the lineages of the two strains were less than 1.5-fold, and the measurements of mixed populations, as in Figure 1B, essentially represent average RTL. If anything the average RTL of older strains would be biased in the direction of shortened telomere lengths, thus making our conclusion that HMEC telomeres are unlikely to shorten significantly with age all the more strongly.

Bottom Line: 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.Telomere 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. MALabarge@lbl.gov.

ABSTRACT

Background: Shortening 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.

Results: RTL 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).

Conclusions: Telomere 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.

No MeSH data available.


Related in: MedlinePlus