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Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Lee JK, Garbe JC, Vrba L, Miyano M, Futscher BW, Stampfer MR, LaBarge MA - Front Cell Dev Biol (2015)

Bottom Line: Cells from all resulting immortal lines exhibited normal karyotypes.Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines.Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA, USA.

ABSTRACT
Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus

Lineage-specific keratin protein expression in non-malignant immortal HMEC on 2-D culture substrata. Representative immunofluorescence images showing keratin (K)14 (red) and K19 (green) expression in (A) 184Dp16sMY, (B) 240Lp16sMY, (C) 240LD1MY, (D) 122Lp16sMY, (E) 122LD1MY, and (F) 805Pp16sMY. Nuclei appear blue, bar represents 50 um.
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Figure 3: Lineage-specific keratin protein expression in non-malignant immortal HMEC on 2-D culture substrata. Representative immunofluorescence images showing keratin (K)14 (red) and K19 (green) expression in (A) 184Dp16sMY, (B) 240Lp16sMY, (C) 240LD1MY, (D) 122Lp16sMY, (E) 122LD1MY, and (F) 805Pp16sMY. Nuclei appear blue, bar represents 50 um.

Mentions: The immortalized lines were evaluated for expression of luminal and myoepithelial/basal lineage-specific proteins and gene transcripts to determine whether their intrinsic subtypes were more luminal- or basal-like. Keratin (K)14 and CALLA/CD10 are proteins expressed primarily in myoepithelia, and K19 and Sialomucin-1/CD227 in luminal epithelia (Villadsen et al., 2007). The two lines derived from the younger women (specimens 184 and 240L) following transduction of p16sh and c-Myc expressed only K14 when grown on 2-D tissue culture plastic (Figures 3A,B). The two lines derived from the older women (specimens 122L and 805P) exhibited mixtures of cells expressing K14, K19, and K14/K19 when exposed to the same p16sh and c-Myc protocol (Figures 3D,F). Both lines derived using cyclin D1/CDK2 to bypass stasis, 240LD1MY from a young woman and 122LD1MY from an older woman, exhibited significant K19 expression, both in cells expressing only K19 as well as expressing K14 and K19 (Figures 3C,E). Flow cytometry (FACS) analysis of CD10 and CD227 expression in normal pre-stasis 240L at 4p shows characteristically distinct CD227+/CD10− luminal and CD227−/CD10+ myoepithelial populations (Figure 4A). All of the p16sMY cell lines had only minor CD227+/CD10− luminal-like populations and generally low CD10 expression, with the exception of 122Lp16sMY that had more CD10 expression relative to the other cell lines (Figure 4B). In contrast, most of the cells of the two D1MY lines were CD227+/CD10+ (Figure 4C).


Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Lee JK, Garbe JC, Vrba L, Miyano M, Futscher BW, Stampfer MR, LaBarge MA - Front Cell Dev Biol (2015)

Lineage-specific keratin protein expression in non-malignant immortal HMEC on 2-D culture substrata. Representative immunofluorescence images showing keratin (K)14 (red) and K19 (green) expression in (A) 184Dp16sMY, (B) 240Lp16sMY, (C) 240LD1MY, (D) 122Lp16sMY, (E) 122LD1MY, and (F) 805Pp16sMY. Nuclei appear blue, bar represents 50 um.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356162&req=5

Figure 3: Lineage-specific keratin protein expression in non-malignant immortal HMEC on 2-D culture substrata. Representative immunofluorescence images showing keratin (K)14 (red) and K19 (green) expression in (A) 184Dp16sMY, (B) 240Lp16sMY, (C) 240LD1MY, (D) 122Lp16sMY, (E) 122LD1MY, and (F) 805Pp16sMY. Nuclei appear blue, bar represents 50 um.
Mentions: The immortalized lines were evaluated for expression of luminal and myoepithelial/basal lineage-specific proteins and gene transcripts to determine whether their intrinsic subtypes were more luminal- or basal-like. Keratin (K)14 and CALLA/CD10 are proteins expressed primarily in myoepithelia, and K19 and Sialomucin-1/CD227 in luminal epithelia (Villadsen et al., 2007). The two lines derived from the younger women (specimens 184 and 240L) following transduction of p16sh and c-Myc expressed only K14 when grown on 2-D tissue culture plastic (Figures 3A,B). The two lines derived from the older women (specimens 122L and 805P) exhibited mixtures of cells expressing K14, K19, and K14/K19 when exposed to the same p16sh and c-Myc protocol (Figures 3D,F). Both lines derived using cyclin D1/CDK2 to bypass stasis, 240LD1MY from a young woman and 122LD1MY from an older woman, exhibited significant K19 expression, both in cells expressing only K19 as well as expressing K14 and K19 (Figures 3C,E). Flow cytometry (FACS) analysis of CD10 and CD227 expression in normal pre-stasis 240L at 4p shows characteristically distinct CD227+/CD10− luminal and CD227−/CD10+ myoepithelial populations (Figure 4A). All of the p16sMY cell lines had only minor CD227+/CD10− luminal-like populations and generally low CD10 expression, with the exception of 122Lp16sMY that had more CD10 expression relative to the other cell lines (Figure 4B). In contrast, most of the cells of the two D1MY lines were CD227+/CD10+ (Figure 4C).

Bottom Line: Cells from all resulting immortal lines exhibited normal karyotypes.Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines.Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA, USA.

ABSTRACT
Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus