Selective segregation of DNA strands persists in long-label-retaining mammary cells during pregnancy - PubMed (original) (raw)

Comparative Study

Selective segregation of DNA strands persists in long-label-retaining mammary cells during pregnancy

Brian W Booth et al. Breast Cancer Res. 2008.

Abstract

Introduction: During pregnancy the mammary epithelial compartment undergoes extreme proliferation and differentiation, facilitated by stem/progenitor cells. Mouse mammary epithelium in nonpregnant mice contains long label-retaining epithelial cells (LREC) that divide asymmetrically and retain their template DNA strands. The role of LREC during alveogenesis has not been determined.

Methods: We performed immunohistochemistry and autoradiography on murine mammary glands that had been labeled with 5-bromodeoxyuridine (5BrdU) during allometric ductal growth to investigate the co-expression of DNA label retention and estrogen receptor-alpha or progesterone receptor during pregnancy. A second DNA label ([3H]-thymidine) was administered during pregnancy to identify label-retaining cells (LRC), which subsequently enter the cell cycle. Use of this methodology allowed us to investigate the co-localization of 5BrdU with smooth muscle actin, CD31, cytokeratin, and desmin in periductal or peri-acinar LRC in mammary tissue from pregnant mice subsequent to a long chase period in order to identify LRC.

Results: Estrogen receptor-alpha positive and progesterone receptor positive cells represented approximately 30% to 40% of the LREC, which is under 1.0% of the epithelial subpopulation. Pregnancy altered the percentage of LREC expressing estrogen receptor-alpha. LRC situated in periductal or peri-acinar positions throughout the gland do not express epithelial, endothelial, or myoepithelial markers, and these undefined LRCs persist throughout pregnancy. Additionally, new cycling LREC ([3H]-thymidine retaining) appear during alveologenesis, and LRC found in other tissue types (for example, endothelium and nerve) within the mammary fat pad become double labeled during pregnancy, which indicates that they may also divide asymmetrically.

Conclusions: Our findings support the premise that there is a subpopulation of LREC in the mouse mammary gland that persists during alveologenesis. These cells react to hormonal cues during pregnancy and enter the cell cycle while continuing to retain, selectively, their original template DNA. In addition, nonepithelial LRC are found in periductal or peri-acinar positions. These LRC also enter the cell cycle during pregnancy. During alveologenesis, newly created label-retaining ([3H]-thymidine) epithelial cells appear within the expanding alveoli and continue to cycle and retain their original template DNA ([3H]-thymidine) strands, as determined by a second pulse of 5BrdU.

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Figures

Figure 1

Figure 1

LRC are in cycle during pregnancy. The nuclear label [3H]-thymidine is detected by autoradiography in sections of mammary tissue from pregnant mice that received 5BrdU for 2 weeks during weeks 4 and 5 of life (group 1 is day 3 of pregnancy, group 2 is day 4 of pregnancy, group 3 is day 6 of pregnancy, and group 4 is day 12 of pregnancy). LRC in periductal and basal positions are evident in all time points evaluated. Arrows indicate double positive cells. Scale bars = 10 μm. 5BrdU, 5-bromodeoxyuridine; LRC, label-retaining cell.

Figure 2

Figure 2

Periductal LRC are not myoepithelial cells. Sections from all experimental groups were stained for the myoepithelial marker SMA (brown). LRC (blue, 5BrdU) in periductal or peri-acinar positions are not SMA positive (arrows). (a) Group 1. (b) Group 2 cells are endothelial except for arrows. (c) Group3. (d) Group 4. Scale bars = 15 μm. 5BrdU, 5-bromodeoxyuridine; LRC, label-retaining cell; SMC, smooth muscle actin.

Figure 3

Figure 3

Periductal LRC are not epithelial cells. Sections from all experimental groups were stained for the epithelial markers cytokeratins (green). LRC (red, 5BrdU) in periductal or peri-acinar positions are not cytokertin positive (arrows). Scale bars = 10 μm. 5BrdU, 5-bromodeoxyuridine; LRC, label-retaining cell.

Figure 4

Figure 4

LRC in blood vessels are CD31 positive. Sections from all experimental groups were stained for the endothelial marker CD31 (brown). LRC (blue, 5BrdU) in blood vessels are also CD31 positive. Scale bars = 10 μm. 5BrdU, 5-bromodeoxyuridine; LRC, label-retaining cell.

Figure 5

Figure 5

LRC in nonepithelial mammary cells. (a) Endothelial cells that line a blood vessel are BrdU positive (brown), and a fraction of those are in cycle, as determined by [3H]-thymidine incorporation (arrow). (b) LRC exist in a mammary nerve (brown, BrdU positive). (c) LRC in fatty stroma (brown, BrdU positive). Scale bars = 10 μm. 5BrdU, 5-bromodeoxyuridine; LRC, label-retaining cell.

Figure 6

Figure 6

Periductal LRC do not have nuclear hormone receptors. Sections from all experimental groups were stained for the nuclear hormone receptors (a) ER-α, (b) PR, or (c) AR (green). Arrows indicate that periductal LRC (red, 5BrdU) are not double positive for the receptors. Scale bars = 10 μm. AR, adrogen receptor; 5BrdU, 5-bromodeoxyuridine; ER, estrogen receptor; LRC, label-retaining cell; PR, progesterone receptor.

Figure 7

Figure 7

Nuclear steroid receptor expressing cells in cycle. The nuclear label [3H]-thymidine is detected by autoradiography in sections from all experimental groups that were also stained for the nuclear steroid receptors (a) ER-α or (b) PR. All experimental groups contained epithelial cells that were ER-α/[3H]-thymidine or PR/[3H]-thymidine double positive. A cell double positive for the nuclear steroid receptor and [3H]-thymidine within the tissue section are displayed in the inset. ER, estrogen receptor; PR, progesterone receptor.

References

    1. Cairns J. Mutation selection and the natural history of cancer. Nature. 1975;255:197–200. doi: 10.1038/255197a0. - DOI - PubMed
    1. Cairns J. Somatic stem cells and the kinetics of mutagenesis and carcinogenesis. Proc Natl Acad Sci USA. 2002;99:10567–10570. doi: 10.1073/pnas.162369899. - DOI - PMC - PubMed
    1. Potten CS, Hume WJ, Reid P, Cairns J. The segregation of DNA in epithelial stem cells. Cell. 1978;15:899–906. doi: 10.1016/0092-8674(78)90274-X. - DOI - PubMed
    1. Potten CS, Owen G, Booth D. Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Sci. 2002;115:2381–2388. - PubMed
    1. Merok JR, Lansita JA, Tunstead JR, Sherley JL. Cosegregation of chromosomes containing immortal DNA strands in cells that cycle with asymmetric stem cell kinetics. Cancer Res. 2002;62:6791–6795. - PubMed

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