The online version of this article (doi:10.1186/bcr2754) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
GHS and CAB conceived the study, the design, and interpreted the data. KMB performed data collection, data interpretation, and wrote the manuscript. CAB and GHS performed the mouse surgeries. DM, FB, and FSK isolated and characterized the immortalized, premalignant COMMA-D cell clones. All authors have read and approved the final manuscript.
During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy.
The glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-α (ER-α) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs).
Cells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups.
The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.
Additional file 1: Supplemental figure S1. LRECs express ER-α and PR and incorporate 3H-TdR into their nuclei. Immunohistochemistry for ER-α and PR, as well as labeling for 3H-TdR, was performed on the intact mammary glands (thoracic) of female mice. Epithelial cells present in the glands expressed ER-α (A-C, red arrow) as well as PR (D-F, red arrow) in the NSP2 (A, D), NSP3 (B, E), and SP3 (C, F) groups. 3H-TdR was incorporated into the nucleus of some cells expressing the steroid receptors (green arrow, inset). Scale bars equal 20 μm. (PDF 2 MB)13058_2010_2741_MOESM1_ESM.PDF
Additional file 2: Supplemental figure S2. Label-retaining cells are present in nonmammary tissues. Cells expressing 5-bromo-2-deoxyuridine were found in (a) cartilage, (b) adipose tissue, (c) skeletal muscle, and (d) periductal cells. Scale bars equal 20 μm. (PDF 9 MB)13058_2010_2741_MOESM2_ESM.PDF
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- Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA
Karen M Bussard
Corinne A Boulanger
Frances S Kittrell
Gilbert H Smith
- BioMed Central
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