Immortalized, pre-malignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA.
BACKGROUND
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.


METHODS
The glands of 3-week old immune competent Balb/C female mice were utilized 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 which retain their template DNA. Nulliparous mice were then either injected with [(3)H]-thymidine ((3)H-TdR) to distinguish 5-BrdU-label retaining cells that enter the cell cycle and euthanized, or mated, injected with (3)H-TdR, and euthanized at various days post-coitus. Sections were stained for estrogen receptor-α(ER-α) or progesterone receptor (PR) via immunohistochemistry. Cells labelled with both 5-BrdU and (3)H-TdR were indicative of label-retaining epithelial cells (LREC).


RESULTS
Cells that retained a 5-BrdU label and cells labelled with [(3)H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labelled nuclei (LREC) were found in the intact mammary gland of both pregnant and nulliparous mice, and in mammary glands implanted with pre-malignant cells. Double-labelled cells ((3)H-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 ((3)H-TdR) to daughter cells; and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary implant groups.


CONCLUSIONS
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.
Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Karen M
KM
Bussard
Corinne A
CA
Boulanger
Frances S
FS
Kittrell
Fariba
F
Behbod
Daniel
D
Medina
Gilbert H
GH
Smith
D000818:Animals; D060049:Asymmetric Cell Division / Q000235:genetics*; D001345:Autoradiography; D001973:Bromodeoxyuridine; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000032:analysis; D005260:Female; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011230:Precancerous Conditions; D011247:Pregnancy; D011980:Receptors, Progesterone / Q000032:analysis; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013698:Templates, Genetic; D013936:Thymidine; D014316:Tritium
D047628:Estrogen Receptor alpha; D011980:Receptors, Progesterone; D014316:Tritium; D004247:DNA; D001973:Bromodeoxyuridine; D013936:Thymidine