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

Abstract

Introduction: 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.

Figure 1

Murine implantation experimental design. The inguinal mammary glands of 3-week-old Balb/C mice were cleared of host epithelium and inoculated with either 20,000 (NSP2) or 50,000 (NSP3, SP3) immortalized, premalignant cells. Control (thoracic) number 3 and 8 glands remained intact. 5-BrdU was administered for 2 consecutive days/week for 5 weeks, followed by a break (chase) period of 3 weeks. Nulliparous female mice were then inoculated with ³H-TdR and subsequently euthanized within 90 minutes. The remaining mice were mated, inoculated with ³H-TdR, and euthanized within 90 minutes of receiving label at either 4 to 6, 8 to 10, or 12 to 15 days after coitus.

Figure 2

Immortal cell populations contain LRECs. Glands and fat pads from nulliparous mice either used intact or implanted with immortalized, premalignant cell clones were labeled with 5-BrdU (green arrow) and ³H-TdR (orange arrow). Double-labeled cells (labeled with both 5-BrdU and ³H-TdR, black arrow, inset) were identified by using light microscopy and were found in both intact glands (a, c, e) and implanted fat pads (b, d, f). LRECs were found in all immortal, premalignant cells (b, NSP2; d, NSP3; f, SP3). Representative images are shown. Scale bars equal 20 μm.

Figure 3

Diagram of symmetric and asymmetric division. Brown background indicates a cell with template DNA; black dots indicate a cell that is cycling. (a) During symmetric division, a cycling (black dots) parent cell with template DNA (brown background) divides equally, yielding two proliferating (black dots) identical daughter cells that each have one template DNA strand (brown background) and one newly synthesized DNA strand. (b) In asymmetric division, a cycling (black dots) parent (stem) cell with template DNA (brown background) undergoes unequal division, retaining its template DNA (brown background), but yielding one proliferating (black dots) daughter cell containing DNA that was newly synthesized from the parent's template strand.

Figure 4

Asymmetric cell division occurred in immortalized, premalignant cell populations. Fat pads from mice implanted with immortalized, premalignant cells were labeled with 5-BrdU and ³H-TdR. Double-labeled cells that undergo asymmetric division and pass a ³H-TdR label on to their daughter cell (red arrow) in (a) nulliparous and (b) pregnant mice were identified by using light microscopy. Scale bars equal 5 μm.

Figure 5

LRECs present in the mammary fat pads of pregnant and nulliparous mice implanted with immortalized, premalignant cell populations express ER-α and PR and incorporate ³H-TdR into their nuclei. Immunohistochemistry for ER-α ((a, c), red arrow) and PR ((b, d), red arrow), and labeling for ³H-TdR, was performed on mouse mammary glands (inguinal) of nulliparous mice (a, b) or pregnant (c, d) implanted with immortalized, premalignant cells. ³H-TdR was incorporated into the nucleus of some cells expressing the steroid receptors (green arrow, inset). Representative images are shown. Scale bars equal 20 μm.

Figure 6

Label-retaining cells were present in immortalized, premalignant cells in vitro. Immortalized, premalignant cell populations NSP2 (a, b), NSP3 (c, d), and SP3 (e, f) were cultured in vitro as described in Materials and methods. Cells were plated, allowed to incubate overnight, treated with 0.5 μM EdU for 24 hours, and fixed, permeabilized, labeled with Alexa Fluor^® 488, and counterstained with DAPI either (a, c, e) immediately or (b, d, f) after serial passage. Representative images are shown. Scale bars equal 40 μm.